Amorphous calcium carbonate for the treatment of calcium malabsorption and metabolic bone disorders

ABSTRACT

Provided are methods for treating calcium malabsorption and conditions associated with calcium malabsorption, employing the administration of a composition containing stable amorphous calcium carbonate. Further provided are methods for increasing bone mineral density in a bone metabolism associated disorders, diseases or conditions, employing the administration of said composition in combination with a bone resorption inhibitor.

FIELD OF THE INVENTION

The present invention relates to natural and synthetic amorphous calciumcarbonate compositions for use in treatment of calcium malabsorption,and malabsorption associated disorders, diseases and conditions, and forincreasing bone mineral density in calcium malabsorption and bonemetabolism associated disorders.

BACKGROUND

Calcium is considered to be one of the most important minerals in thehuman body. It is required for maintaining bone mineral density, isessential for exocytosis of neurotransmitters, takes part in thecontraction of muscle cells, replaces sodium as the depolarizing mineralin the heart, and participates in many other physiological functions.Calcium gastrointestinal absorption depends not only on the dietarycalcium availability but on the absorptive capacity of the intestines,which is affected by physiological factors such as calcium reserves,hormonal regulation or previous dietary calcium supply. Dissolution ofcalcium salts (e.g. calcium carbonate) in the stomach is one step in theproper active and passive absorption of calcium as a calcium ion(Ca⁽²⁺⁾) in the proximal small intestine. Stomach acid markedlyincreases dissolution and ionization of poorly soluble calcium salts. Ifacid is not properly secreted, calcium salts are minimally dissolved(ionized) and, subsequently, may not be properly and effectivelyabsorbed. Atrophic gastritis, gastric surgery, and high-dose, long-termuse of antisecretory drugs markedly reduce acid secretion and may,therefore, be risk conditions for malabsorption of dietary andsupplementary calcium, and may thereby increase the risk of osteoporosisin the long term (Sipponen et al., Scand J Gastroenterol. 2010; 45(2):133-8).

Calcium gastrointestinal absorption may also be decreased in patientsafter bariatric surgery, patients suffering from hypoparathyroidism,Crohn's disease, cystic fibrosis, inflammatory bowel disease or celiacdisease. Individuals, consuming additional types of drugs, such asproton pump inhibitors, anticonvulsants, and chronic corticosteroids,may also develop calcium malabsorption.

Bioavailability of calcium depends on its gastrointestinal absorptionand the incorporation of absorbed calcium into bone. As for intestinalabsorption, physiological factors, particularly hormones, play a majorrole in the incorporation of calcium into bone. The bioavailability ofcalcium may therefore be defined as the fraction of dietary calcium thatis potentially absorbable by the intestine and can be used forphysiological functions, particularly bone mineralization, or to limitbone loss (Gueguen et al, J Am Coil Nutr, 2000 vol. 19 no. suppl 2119S-136S).

Bone mineral density loss is associated with various metabolic bonediseases, such as: osteopenia, osteomalacia, Rickets, osteitis fibrosacystica, and osteoporosis. Studies have shown that inadequate intake ofdietary calcium can induce many bone-related diseases, such asosteoporosis.

A standard medication for prevention and treatment of certain types ofbone loss (including osteoporosis) is an anti-resorptive agent. Onenon-limiting example for the anti-resorptive agents are bisphosphonates,e.g. the bisphosphonate Alendronate (ALN). Administration of ALNattenuates the decline in bone mineral density (BMD), as ALN has a boneresorption inhibiting effect. However, it is an acknowledged problemthat ALN also suppresses bone formation and its administration isassociated with a risk of adverse symptoms. Use of several drugs incombination has been suggested for the improvement of patients'compliance and the therapeutic effect.

The calcium used in supplements today, whether obtained from naturalsources or synthetic precipitates, may comprise both organic andinorganic calcium salts. In specific conditions where calciumgastrointestinal absorption is limited, standard intake of the availablesupplements is insufficient to promote absorption, resulting in a needto increase intake doses. The requirement of consumption of highercalcium doses in malabsorption-associated conditions leads to adverseeffects like constipation, kidney stones, vascular problems andsubsequent reduced compliance. Moreover, most calcium supplementsrequire low gastric pH in order to be efficiently dissolved and absorbedthrough the gut, and thus their bioavailability is superior in a fastingstate.

Over the past 20 years, a rapidly growing scientific interest in thethermodynamically unstable amorphous polymorph of calcium carbonate,named amorphous calcium carbonate (ACC), has emerged. In nature, ACC isutilized by a small number of organisms, mainly crustaceans and otherinvertebrates that developed capabilities for stabilizing ACC intransient mineral deposition sites. These organisms require anexceptional efficient mineral source for the periodical mobilization,absorption and precipitation of calcium. In some crustaceans, such asthe freshwater crayfish, ACC is stored in large quantities inspecialized transient storage organs, named the Gastrolith.

In recent years, some of the inventors of the present invention havedisclosed use of the gastrolith organs, ground to a fine powder usefulas pharmaceutical and nutraceutical calcium compositions (WO 05/115414).It was disclosed that daily oral consumption of compositions comprisinggastrolith components dramatically improves a range of conditions suchas bone disorders, bone fractures, and cancer (WO 2008/041236).Pharmaceutical and nutraceutical compositions comprising ACC andphosphorylated peptides or amino acids for treating various disordersand conditions are disclosed in WO 2009/053967.

There is an unmet need for efficient treatment of calcium malabsorption,calcium malabsorption associated bone density loss and bone metabolismassociated diseases.

SUMMARY OF THE INVENTION

The present invention provides a method of use of a compositioncomprising synthetic or natural stable ACC for treatment of calciummalabsorption. It is disclosed herein for the first time that ACC canovercome the deficiencies of other types of calcium supplements even inpatients suffering from calcium malabsorption.

The treatment of calcium malabsorption may furthermore prevent ordecrease or delay the onset of calcium deficiency related disorders,diseases. According to some embodiments conditions related to calciumdeficiency include patients after bariatric or gastric surgery, patientssuffering from hypoparathyroidism, vitamin D deficiency, renal tubulardiseases, renal failure, pancreatitis, hypoproteinemia, andhyperphosphatemia. According to alternative embodiments diseasesinvolving calcium malabsorption include Crohn's disease, cysticfibrosis, inflammatory bowel disease, celiac disease, and atopicgastritis. According to further embodiments calcium malabsorption mayencompass subjects with enhanced bone formation and individualsconsuming corticosteroids, proton pump inhibitors, anticonvulsants,rifampin and similar antibiotics, chelating agents, and antisecretorydrugs.

Enhanced calcium gastrointestinal absorption of stable ACC compared toother calcium supplements may delay or minimize conditions and disordersresulting from calcium deficiency in other populations includingpostmenopausal women, and perimenopausal women, elderly men, children,adolescents, pregnant women, and breastfeeding women.

Enhanced calcium bioavailability of ACC may further delay or minimizebone density loss in subjects suffering from calcium malabsorption. Thepresent invention provides use of a composition comprising synthetic ornatural stable ACC for increasing the bone mineral density in a subjectin metabolic bone disorders, diseases and conditions. The composition ofthe present invention is further used for delaying the onset and fortreatment of said disorders, diseases or conditions, comprisingosteomalacia, Paget's disease of bone, osteopenia, osteitis fibrosacystica, Rickets, osteoporosis and acute alcohol consumption

In one aspect, the invention provides a method of treating calciummalabsorption in a subject in need of such treatment, comprisingadministering to said subject an effective amount of stable amorphouscalcium carbonate (ACC) comprising at least one stabilizer. According tosome embodiments ACC is of synthetic origin. According to otherembodiments ACC is of natural origin.

According to some embodiments the stabilizer is selected from the groupconsisting of organic acids, phosphoric or sulfuric esters of hydroxycarboxylic acids, hydroxyl bearing organic compounds, and combinationsthereof. Each possibility represents a separate embodiment of theinvention. In some embodiments, said stabilizer comprises at least onecomponent selected from phosphoric or sulfuric esters of hydroxylcarboxylic acids and hydroxyl bearing organic compounds. In someembodiments, said stabilizer comprises at least one component selectedfrom phosphorylated amino acids and polyols. Said amino acids may bepresent in amino acid derivatives or oligopeptides or polypeptides, andsaid polyols may comprise alcohols or saccharides. Each possibilityrepresents a separate embodiment of the invention. According to someembodiments the phosphorylated amino acids are selected fromphosphoserine and phospho-threonine. According to some embodiments, saidstabilizer comprises at least one saccharide selected from mono-, di-,oligo-, and polysaccharides. According to some embodiments, saidstabilizer comprises hydroxyl bearing organic compounds further combinedwith at least one alkali hydroxide. According to some embodiments, thestabilizer is a carboxylic acid, preferably citric acid, tartaric acidor malic acid. In some embodiments, said stabilizer comprises at leastone compound selected from phosphorylated amino acids, phosphorylatedpeptides, chitin with at least one peptide, and polyol with alkalinehydroxide. According to some exemplary embodiments, said ACC is obtainedfrom isolated crustacean gastroliths. According to one embodiment, saidnatural ACC is stabilized by chitin and polypeptides. According toanother embodiment, the ACC is synthetic, wherein said synthetic ACC isstabilized by phosphorylated amino acids selected from phosphoserine orphosphothreonine. According to another embodiment said synthetic ACC isstabilized by phosphoserine in combination with citric acid. Accordingto still another embodiment, said synthetic ACC is stabilized by citricacid. According to yet another embodiment, said ACC is stabilized bysucrose in combination with sodium hydroxide.

According to some embodiments, the subject suffers from a disorder ofcalcium metabolism associated with a decrease of plasma calciumconcentration below 8.8 mg/dL. According to some embodiments, thesubject in need of the treatment is selected from the group consistingof patients suffering from hypoparathyroidism, vitamin D deficiency,renal tubular diseases, renal failure, pancreatitis, hypoproteinemia,Crohn's disease, cystic fibrosis, inflammatory bowel disease, celiacdisease, hyperphosphatemia, atopic gastritis, patients after bariatricor gastric surgery, subjects with enhanced bone formation, and subjectsobtaining medicaments selected from corticosteroids, proton pumpinhibitors, anticonvulsants, rifampin and similar antibiotics, chelatingagents, and antisecretory drugs. According to some embodiments, thesubject is selected from postmenopausal or perimenopausal women.According to some embodiments said subject is susceptible to thedevelopment of bone mineral density loss associated disorders, diseasesand conditions. The bone density loss associated disorder in a subjectin a need of calcium malabsorption treatment, may be osteoporosis.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising stable amorphous calcium carbonate (ACC)comprising at least one stabilizer selected from the group consisting oforganic acids, phosphoric or sulfuric esters of hydroxy carboxylicacids, and hydroxyl bearing organic compounds, for use in treatingcalcium malabsorption. The pharmaceutical composition according to theinvention may further comprise carriers, adjuvants, diluents, orexcipients.

In another embodiment, the invention is directed to the use of amorphouscalcium carbonate, comprising at least one stabilizer selected from thegroup consisting of organic acids, phosphoric or sulfuric esters ofhydroxy carboxylic acids, and hydroxyl bearing organic compounds fortreating calcium malabsorption.

In another embodiment, the invention is directed to the use of amorphouscalcium carbonate, comprising at least one stabilizer selected from thegroup consisting of organic acids, phosphoric or sulfuric esters ofhydroxy carboxylic acids, and hydroxyl bearing organic compounds in thepreparation of a medicament for treating calcium malabsorption.

In another aspect, the invention relates to a composition comprisingstable amorphous calcium carbonate (ACC) comprising at least onestabilizer for the treatment of calcium malabsorption associateddisorders, diseases and conditions.

The invention further encompasses the use of a composition comprisingstable amorphous calcium carbonate (ACC) in the preparation of amedicament for treatment (in fed or fasting state) of calciummalabsorption in calcium malabsorption associated disorders, diseasesand conditions, wherein the ACC is finely mixed with at least onestabilizer. In one embodiment composition comprising stable ACC has asuperior gastrointestinal absorption when administered in a fed state.In another embodiment composition comprising stable ACC has a superiorgastrointestinal absorption when administered in a fasting state.

In another aspect, the invention provides a method for the enhancementof bone mineral density in a bone metabolism associated disorder,disease or condition, comprising administering an effective amount of acomposition comprising stable amorphous calcium carbonate (ACC) and atleast one stabilizer to a mammalian subject. According to someembodiments ACC is of synthetic origin. According to other embodimentsACC is of natural origin. According to some embodiments, the stabilizeris selected from the group consisting of organic acids, phosphoric orsulfuric esters of hydroxy carboxylic acids, hydroxyl bearing organiccompounds, and combinations thereof. Each possibility represents aseparate embodiment of the invention. In some embodiments, saidstabilizer comprises at least one component selected from phosphoric orsulfuric esters of hydroxyl carboxylic acids and hydroxyl bearingorganic compounds. In some embodiments, said stabilizer comprises atleast one component selected from phosphorylated amino acids andpolyols. Said amino acids may be present in amino acid derivatives oroligopeptides or polypeptides, and said polyols may comprise alcohols orsaccharides. Each possibility represents a separate embodiment of theinvention. According to some embodiments, the phosphorylated amino acidsare selected from phosphoserine and phosphothreonine. According to someembodiments, said stabilizer comprises at least one saccharide selectedfrom mono-, di-, oligo-, and polysaccharides. According to someembodiments, said stabilizer comprises hydroxyl bearing organiccompounds further combined with at least one alkali hydroxide. Accordingto some embodiments, the stabilizer is a carboxylic acid, preferablycitric acid, tartaric acid or malic acid. In some embodiments, saidstabilizer comprises at least one compound selected from phosphorylatedamino acids, phosphorylated peptides, chitin with at least one peptide,and polyol with alkaline hydroxide. According to some embodiments, saidACC is obtained from isolated crustacean gastrolith.

According to some embodiments, the method for the enhancement of bonemineral density comprises administration of the composition comprisingstable ACC in combination with a bone resorption inhibitor. According tosome embodiments, the bone resorption inhibitor is bisphosphonate.According to some embodiments, the bisphosphonate is selected fromAlendronate, Risedronate, Tiludronate, Ibandronate, Zolendronate,Pamidronate, Etidronate, and salts and esters thereof. According to oneembodiment, the bisphosphonate is Alendronate. In some embodiments, thecomposition is administered in combination with lower therapeutic dosesof bisphosphonate Alendronate, compared to the dose required withoutcalcium supplements or with calcium supplements other than stable ACCsupplements. According to some embodiments, the invention provides amethod for the enhancement of bone mineral density in bone metabolismassociated disorders, diseases or conditions, selected fromosteoporosis, oseomalacia, Paget's disease of bone, osteopenia, osteitisfibrosa cystica and Rickets. In one specific embodiment, the inventionprovides a method for enhancing bone mineral density in osteoporosis.According to some embodiments, the invention provides a method for theenhancement of bone mineral density in bone metabolism associateddisorders, diseases or conditions, selected from osteomalacia, Paget'sdisease of bone, osteopenia, osteitis fibrosa cystica, Rickets,osteoporosis and acute alcohol consumption, comprising administration ofsaid composition in combination with bone resorption inhibitor. In onespecific embodiment, the invention provides a method for enhancing bonemineral density in osteoporosis, comprising administration of saidcomposition in combination with bisphosphonate Alendronate. According tothe method of the present invention, the composition is administered toa mammalian subject selected from postmenopausal women, elderly men,children, adolescents, pregnant women, and breastfeeding women.According to some embodiments, the method for the enhancement of bonemineral density comprises administration of the composition comprisingstable ACC, wherein said subject is selected from patients sufferingfrom calcium malabsorption and calcium malabsorption associateddisorders, diseases and conditions.

The invention further provides the use of a composition comprisingstable amorphous calcium carbonate (ACC) comprising at least onestabilizer, selected from the group consisting of organic acids,phosphoric or sulfuric esters of hydroxy carboxylic acids, and hydroxylbearing organic compounds for use in the preparation of a medicament forthe enhancement of bone mineral density in a bone metabolism associateddisorder, disease or condition The pharmaceutical composition accordingto the invention may further comprise carriers, adjuvants, diluents, orexcipients.

In still another aspect, the invention provides a method of enhancingcalcium gastrointestinal absorption in a subject suffering from calciummalabsorption, comprising administering to said subject an effectiveamount of ACC comprising at least one stabilizer. According to someembodiments, the administration is performed to said subject selectedfrom a subject in a fasting state and in a fed state.

All the above and other characteristics and advantages of the inventionwill be further understood through the following illustrative andnon-limitative description of embodiments thereof, with reference to theappended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Calcium levels are elevated following amorphous calciumcarbonate (ACC) vs. crystalline calcium carbonate administration to rats

Results are means±SEM. Student's t-test: *p<0.05

FIG. 2: Calcium fractional absorption is increased by 2.1 fold per eachwoman following amorphous calcium carbonate administration (ACC) vs.crystalline calcium carbonate (CCC) in fed postmenopausal women

Paired t-test: p<0.01

FIG. 3: Calcium fractional absorption is increased by 1.9 fold as twoseparated groups analyzed following amorphous calcium carbonateadministration (ACC) vs. crystalline calcium carbonate (CCC) in fedpostmenopausal women

Results are means±SEM. Student's t-test: *p<0.05

FIG. 4: Calcium fractional absorption is increased by 4.6 followingamorphous calcium carbonate administration (ACC) vs. crystalline calciumcarbonate (CCC) in one fasted postmenopausal woman

FIGS. 5A-5E: Three-dimensional reconstruction of a representative distalfemurs and 4^(th) vertebras cross sections from each treatment group.

Tubercular bone region for Sham (FIG. 5A); Crystalline calcium carbonate(CCC) (FIG. 5B); amorphous calcium carbonate (ACC) (FIG. 5C);CCC+alendronate (ALN) (FIG. 5D); and ACC+ALN (FIG. 5E). Scale barrepresents 1.5 mm distance.

FIGS. 6A-6D: Trabecular bone mineral density (Tb.BMD) and bone volumefrom total bone tissue volume (BV/TV) of the groups obtained by KT.

FIG. 6A: Tb.BMD of the distal femur.

FIG. 6B: Tb.BMD of the 4th lumbar vertebra.

FIG. 6C: BV/TV of the distal femur.

FIG. 6D: BV/TV of the 4th lumbar vertebra.

Percentage represent increase from CCC treated group (control).

FIGS. 7A-7E: Three-dimensional reconstruction of a representative distalfemurs and 4^(th) vertebras cross sections from each treatment group.

Tubercular bone region for sham (FIG. 7A); control (FIG. 7B); gastrolith(Gast) (FIG. 7C); amorphous calcium carbonate (ACC) (FIG. 7D); andcitrate (FIG. 7E). Scale bar represents 1.5 mm distance.

FIGS. 8A-8B: Trabecular bone mineral density (Tr.BMD) of the groupsobtained by μCT.

FIG. 8A: Tr.BMD of the distal femurs.

FIG. 8B: Tr.BMD of the 4^(th) lumbar vertebras.

One-way ANOVA: p<0.001. Letters represent Fishers LSD post-hoccomparison.

FIGS. 9A-9B: Trabecular bone volume from total bone tissue (BV/TV) ofthe treatment groups obtained by μCT.

FIG. 9A: BV/TV of the distal femurs.

FIG. 9B: BV/TV of the 4th lumbar vertebras.

One-way ANOVA: p<0.001. Letters represent Fishers LSD post-hoccomparison.

FIGS. 10A-10G: Mechanical microarchitectural properties of the lumbarvertebras.

Ultimate Force (FIG. 10A); Energy to ultimate force (UF) (FIG. 10B);Toughness (FIG. 10C); Energy to yield (FIG. 10D); Trabecular bonepattern factor (TBPf) (FIG. 10E); Structure model index (SMI) (FIG.10F); Degree of anisotropy (DA) (FIG. 10G).

One-way ANOVA: p<0.01. Letters represent Fishers LSD post-hoccomparison. Bars represent SEM.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a previously unknown use of compositionscomprising stable amorphous calcium carbonate (ACC). The amorphouscalcium carbonate based compositions according to the present inventionwere found to be superior calcium sources over commonly marketed calciumsupplements. It has now been found that a composition comprising ACC hasa surprisingly high calcium bioavailability, comprising higherabsorbability in the gastrointestinal tract and in the bones. Thus theuse of ACC is beneficial in calcium malabsorption associated disorders,diseases and conditions (both in fed and even greater advantage infasting state) and in bone loss related disorders and conditions.

The term “treatment” as employed herein refers to the administration ofACC and enhancement of calcium absorption in gastrointestinal tract andin bones.

The terms “delaying the onset” or “prophylaxis” as employed herein referto the postponement of development of the bone diseases, postponement ofdevelopment of symptoms and/or a reduction in the severity of suchsymptoms that will or are expected to develop.

The term “stable ACC” is used herein to indicate that the calciumcarbonate is maintained in the amorphous state for long periods of time,e.g., from several weeks to several years, with no more than 5%conversion into the crystalline form over the said period of time. Thecrystallization of the ACC is inhibited according to the invention bythe addition of one or more stabilizer, selected from organic orinorganic ingredients such as phosphorylated amino acids, other organicacids, peptides, salts, saccharides, or lipids. Said salts may comprise,for example, cations selected from magnesium, potassium, strontium, andsodium, and anions selected from carbonate, phosphate, sulfate,chloride, bromide, fluoride, citrate, fumarate, malate or other organicanions; the terms anion and cation are used to simply describe the saltcomposition, without implying anything about the solubility or pH of thesaid molecules.

The terms “stabilizer” or “stabilizing agent” as used herein are usedinterchangeably and refer to any substance that preserves calciumcarbonate in the amorphous state.

The term “calcium malabsorption” as used herein refers to abnormality inabsorption of dietary calcium across the gastrointestinal tract. Theterm “bone loss” as used herein refers to any decrease in bone cells ortissue, decrease in bone mass, decrease of bone minerals, or decrease ofbone mineral density in a subject.

The term “effective amount” as used herein refers to a sufficient amountof the compositions of comprising stable ACC to treat calciummalabsorption, calcium malabsorption and mineral bone density associateddiseases, disorders and conditions and to enhance mineral bone densityat a reasonable benefit/risk ratio applicable to any medical ornutritional treatment.

In one embodiment, the present invention provides a synthetic(artificial) composition comprising stable ACC and an amount of astabilizer sufficient to maintain the ACC in a non-crystalline state.The stabilizer is selected from, but not limited to, organic acids,phosphoric or sulfuric esters of hydroxyl carboxylic acids and hydroxylbearing organic compounds. According to some embodiments, saidstabilizer comprises at least one component selected from phosphoric orsulfuric esters of hydroxyl carboxylic acids, such asphosphoenolpyruvate, phosphoserine, phosphothreonine, sulfoserine orsulfothreonine and hydroxyl bearing organic compounds, selected frommono-, di-, tri-, oligo- and poly-saccharides, for example, sucrose,mannose, glucose. The stabilizer comprising hydroxyl bearing compoundmay further comprise at least one alkali hydroxide, such as sodiumhydroxide, potassium hydroxide and the like. In some embodiments of theinvention, said stabilizer is selected from phosphorylated amino acidsand polyols. The phosphorylated acids may be present in oligopeptidesand polypeptides. In other embodiments of the invention, the stabilizeris an organic acid, preferably a carboxylic acid. The carboxylic acid ispreferably selected from citric acid, tartaric acid or malic acid.

In one embodiment of the invention, the ACC is stabilized byphosphoserine (P-Ser) or phosphothreonine (P-Thr). In anotherembodiment, the stable ACC comprises a combination of sucrose and sodiumhydroxide. In still another embodiment of the invention, the ACC isstabilized by citric acid. In yet another embodiment of the invention,the ACC is stabilized by a combination of phosphoserine and citric acid.In a preferred embodiment of the invention, an artificial compositioncomprising stable ACC comprising traces of one or more stabilizers isadministered to a person in need of increased or improved calciumabsorption.

In another embodiment, the present invention provides a naturalcomposition, comprising stable ACC present in isolated crayfishgastrolith. The ground gastrolith comprises ACC, organic matterconsisting mainly of chitin and polypeptides, and salts. The componentspresent in the organic matter stabilize the ACC and prevent itscrystallization.

In one aspect, the high calcium bioavailability presented in theinventions is not related to the stabilizing molecule. In anotheraspect, the present invention claims that the high calciumbioavailability is only related to the amorphous state of the calciumcarbonate.

In another aspect, the present invention provides a compositioncomprising stable ACC, comprising at least one stabilizer for use intreatment of calcium malabsorption in patients suffering from calciummalabsorption and calcium malabsorption associated disorders, diseasesand conditions. In another aspect, there is provided a method for theenhancement of calcium gastrointestinal absorption in a subjectsuffering from calcium malabsorption, comprising administering to saidsubject an effective amount of a composition comprising stable amorphouscalcium carbonate, comprising at least one stabilizer.

According to some embodiments conditions related to calcium deficiencyinclude patients after bariatric or gastric surgery, patients sufferingfrom hypoparathyroidism, vitamin D deficiency, renal tubular diseases,renal failure, pancreatitis, hypoproteinemia, and hyperphosphatemia.According to alternative embodiments diseases involving calciummalabsorption include Crohn's disease, cystic fibrosis, inflammatorybowel disease, celiac disease, and atopic gastritis. According tofurther embodiments calcium malabsorption may encompass subjects withenhanced bone formation and individuals consuming corticosteroids,proton pump inhibitors, anticonvulsants, rifampin and similarantibiotics, chelating agents, and antisecretory drugs.

Reduced calcium gastrointestinal absorption may further lead to bonemineral density loss. In another aspect, the present invention providescompositions comprising stable ACC, comprising at least one stabilizerfor use in increasing mineral bone density in patients suffering fromcalcium malabsorption associated disorders, diseases and conditions. Thepresent invention further provides a composition comprising stable ACCcomprising at least one stabilizer for treatment of bone density loss ina subject suffering from calcium malabsorption. Enhanced calciumgastrointestinal absorption of stable ACC compared to other calciumsupplements may delay or minimize conditions and disorders resultingfrom calcium deficiency in populations including postmenopausal women,and perimenopausal women, elderly men, children, adolescents, pregnantwomen, and breastfeeding women.

In yet another aspect, the present invention provides a compositioncomprising stable ACC comprising at least one stabilizer for use inenhancement of bone mineral density in bone metabolism associateddisorders, diseases or conditions. According to one embodiment of theinvention, the bone metabolism associated disorders, diseases andconditions are selected from oseomalacia, Paget's disease of bone,osteopenia, osteitis fibrosa cystica, Rickets, osteoporosis and acutealcohol consumption. According to some embodiments, said composition maybe administered in combination with other medications for prevention andtreatment of bone loss. The medications for prevention and treatment ofbone loss are selected from bone resorption inhibitors, comprisingbisphosphonates (salts of bisphosphonic acid), estrogen receptormodulators, androgen receptor modulators, calcitonin formulations,alpha-calcitonin gene-related peptide formulations, ipriflavoneformulations, anabolic steroid formulations, anti-RANKL (receptoractivator of NF-kappa B ligand) antibody and the like. One non-limitingexample of a bisphosphonate used for prevention or treatment of bonemineral density loss is the bisphosphonate Alendronate. The use of acomposition comprising ACC in combination with bone resorptioninhibitors provides an additive effect on increasing bone mineraldensity. Without wishing to be bound by any specific theory, theadditive effect provided by ACC may be attributed to the highbioavailability and bone-formation inducing effect thereof.Administration of ACC in combination with bone resorption inhibitorallows the reduction of therapeutic doses of bone resorptive inhibitordue to the observed additive effect, as compared to administration ofALN in combination with crystalline calcium carbonate. In someembodiments, the invention provides a method for enhancing bone mineraldensity in bone metabolism associated disorders, comprisingadministering a composition comprising stable ACC comprising at leastone stabilizer in combination with bisphosphonates. In the context ofthe present invention the term combination therapy encompassesadministration of two or more active ingredients in a single dosage formor in separate dosage forms. Separate dosage forms may be administeredsimultaneously or sequentially or on entirely independent separateregimens. For example, the ACC may be administered daily and thebisphosphonate may be administered less frequently.

In some embodiments, said composition is administered in combinationwith lower therapeutical doses of bisphosphonate, compared to the doserequired without calcium supplements or with calcium supplements otherthan stable ACC supplements e. In some embodiments, the bisphosphonateis Alendronate. In some embodiments, said composition is administered incombination with lower therapeutical doses of Alendronate, compared tothe dose required without calcium supplements or with calciumsupplements other than stable ACC supplements. In some embodiments, saidcomposition in combination with ALN is used for enhancing bone mineraldensity in osteoporosis.

In yet another aspect, the present invention provides a compositioncomprising stable ACC, comprising at least one stabilizer for treatmentof bone mineral density loss-associated disorders. According to oneembodiment of the invention, the bone mineral density loss-associateddisorders, diseases and conditions are selected from osteomalacia,osteopenia, osteitis fibrosa cystica, Rickets, osteoporosis and acutealcohol consumption.

In still another aspect, the present invention provides a compositioncomprising stable ACC, comprising at least one stabilizer for delayingthe onset or prophylaxis of bone mineral density loss-associateddisorders. According to one embodiment of the invention, the bonemineral density loss-associated disorders, diseases and conditions areselected from osteomalacia, osteopenia, osteitis fibrosa cystica,Rickets, osteoporosis and acute alcohol consumption.

According to the method of the present invention, the compositioncomprising stable ACC is particularly advantageous for use in subjectssusceptible to decrease in bone mineral density or to the development ofa bone metabolism associated disorder, such as postmenopausal women andelderly men. In another aspect, the compositions of the invention areadministered to subjects required to consume high levels of calcium suchas children, adolescents and women during pregnancy and breastfeeding.In yet another aspect, the compositions of the invention areadministered to subjects suffering from calcium malabsorption.

In one aspect, the present invention relates to the use of compositionscomprising stable ACC comprising at least one stabilizer in thepreparation of medicaments for treatment of calcium malabsorption inpatients suffering from calcium malabsorption associated disorders,diseases and conditions.

In a still further aspect, the present invention relates to the use of acomposition comprising stable ACC comprising at least one stabilizer inthe preparation of medicaments for enhancement of bone mineral densityin a bone metabolism associated disorder, disease or condition. In someembodiments said medicaments are used in combination with boneresorption inhibitors. In a yet further aspect, said compositions areused in the preparation of medicine for treatment of bone mineraldensity loss associated diseases, disorders and conditions. In a stillfurther aspect, the present invention relates to the use of acomposition comprising ACC in the preparation of medicaments forprophylaxis of bone mineral density loss associated diseases, disordersand conditions and calcium malabsorption related conditions. In a stillfurther aspect, the compositions of the present invention are used forincreasing calcium absorption in populations susceptible to thedevelopment of metabolic bone disorders, diseases and conditions. In oneembodiment, the present invention relates to the oral administration ofa composition comprising stable ACC having a superior gastrointestinalbioavailability when administered in a fed state. In another embodiment,the present invention relates to the oral administration of acomposition comprising stable ACC in a fasting state to reach a greaterefficient gastrointestinal bioavailability.

Evidence has been provided, supporting the notion that it is theamorphous calcium carbonate alone, regardless of the stabilizingmolecule or mechanism, that promotes the higher bioavailability ofcalcium when administered as ACC. In an important embodiment of theinvention, synthetic ACC has a superior gastrointestinal bioavailabilitywhen administered in a fed state. In another important embodiment of theinvention, synthetic ACC has a superior gastric bioavailability whenadministered in a fasting state. In a still other important embodimentof the invention, ACC from natural sources has a superior gastricbioavailability when administered in a fed state. In a further importantembodiment of present invention, ACC from natural sources has a superiorgastric bioavailability when administered in a fasting state. Accordingto some embodiments, the administration of stable ACC comprising atleast one stabilizer to a subject in need of such treatment is performedregardless of whether the subject is in a fasting state or in a fedstate.

The compositions of the invention may be preferably administered orallyin various oral forms including, but not limited to, tablets, capsules,pills, powders, granules, elixirs, tinctures, suspensions, syrups,emulsions and as gel form.

In instances in which oral administration is in the form of a tablet orcapsule, the composition components can be combined with a non-toxicpharmaceutically acceptable inert carrier or excipients such as lactose,starch, sucrose, glucose, modified sugars, modified starches,methylcellulose and its derivatives, mannitol, sorbitol, and otherreducing and non-reducing sugars, magnesium stearate, stearic acid,sodium stearyl fumarate, glyceryl behenate, amorphous silica gel orother desiccant material and the like.

The compositions of the invention may be administered in daily doses offrom 0.5 to about 5 g. According to alternative embodiments, thecompositions of the invention may be administered in daily doses of from1.5 to about 20 g. The compositions of the invention may be administeredin daily doses comprising elemental calcium in a range of from 0.15 toabout 1.5 g. According to alternative embodiments, the compositions ofthe invention may typically be administered in daily doses comprisingelemental calcium in a range of from 0.5 to about 6 g.

For oral administration in liquid form, the composition components canbe combined with non-toxic pharmaceutically acceptable inert carrierssuch as ethanol, glycerol, water and the like. When desired or required,suitable binders, lubricants, disintegrating agents and coloring andflavoring agents can also be incorporated into the mixture.

A particular advantage of the compositions according to the invention istheir confirmed low toxicity and high safety for oral administration.Accordingly, the compositions of the invention are, in other aspect ofthe invention, advantageously used as medical foods.

The inventors have conducted experiments aiming to evaluate thebioavailability of calcium source comprising stable ACC. Thebioavailability of calcium from ACC was evaluated both in terms ofcalcium gastrointestinal absorption and of calcium availability to bonemineralization process (bone absorbability). The gastrointestinalabsorption of calcium in generally healthy population and in populationsuffering or susceptible to suffering from calcium malabsorption wasassessed. Reduced calcium gastrointestinal absorption is one of thecauses of bone density loss. Therefore, calcium bone absorbability inpopulations suffering from calcium malabsorption was also assessed. Theuse of stable ACC may be beneficial for enhancing calciumgastrointestinal absorption and for increasing bone mineral density insubjects suffering or susceptible to suffering from calciummalabsorption. Calcium gastrointestinal absorption was further evaluatedin populations susceptible to the bone loss-related disorders andconditions, e.g. postmenopausal women.

The enhanced calcium availability to bone mineralization process wasfound to increase bone mineral density and to positively affect otherbone parameters. As exemplified hereinbelow, the effect of stable ACCcomprising different stabilizers on various bone parameters wasevaluated. The effect of ACC on bone mineral density and other boneparameters in the osteoporotic rat model was also evaluated. Amorphouscalcium carbonate based compositions were also combined with boneanti-resorptive medications, such as Alendronate bisphosphonate andtheir mutual effect on bone fraction parameters was evaluated.

The bioavailability measurements comprised the evaluation of calciumabsorption from stable ACC source in the gastrointestinal tract by meansof serum and urine sampling, based on widely reported bioavailabilitymodels used for the assessment of various drugs and supplements, and theevaluation of the effect of calcium from stable ACC source on bonemineral density and other bone parameters, based on the widely reportedosteoporosis prevention and treatment models, used for the assessment ofvarious drugs and supplements. The bioavailability of stable ACC wasevaluated in patients suffering from malabsorption, e.g. patientssuffering from hypoparathyroidism and individuals consumingcorticosteroids and in populations suffering from bone mineral densityassociated disorders. The enhanced bioavailability of calcium from theACC source allows use of ACC for treatment and prophylaxis of variouscalcium malabsorption and metabolic bone associated diseases, disordersand conditions alone or in combination with standard medications fortreatment of bone loss. Having now generally described the invention,the same will be more readily understood through reference to thefollowing examples, which are provided by way of illustration and arenot intended to be limiting of the present invention.

EXAMPLES

Trial 1: Gastrointestinal Absorption of Calcium from ACC Source.

The objective of the present experiment was to evaluate gastrointestinalabsorption of the calcium source comprising stable Amorphous CalciumCarbonate (ACC) in the rat radioisotope labeling model.

Twenty-six 2 month-old male Wistar rats, weighing 240±15 g, were orallyadministered with a single gelatin capsule containing either ACC(amorphous calcium carbonate) or CCC (crystalline calcium carbonate)intrinsically labeled with ⁴⁵Ca, followed by measurements of calciumfractional absorption in serum and calcium excretion.

FIG. 1 presents the changes in serum calcium concentration, ascalculated by the radioactive readings normalized to the administereddose. The C_(max) values in rats that received ACC were significantlyhigher (up to 40%) than those in the CCC group (FIG. 1 and Table 1).

TABLE 1 Pharmacokinetic parameters of calcium in the serum followingoral administration of radioactive calcium carbonate preparationsCompound C_(max) (μg/mL) T_(max) (h) AUC_(∞)(μg × h/mL) CCC 5.8 ± 0.07^(a) 3.0 ± 0.3 ^(a) 109.7 ± 6.4 ^(a) ACC 8.1 ± 0.8 ^(b)  2.8 ± 0.2 ^(a)134.5 ± 7.0 ^(b) Different superscript letters represent statisticalsignificance (p < 0.05), as determined by ANOVA

Pharmacokinetic analysis indicates that the gastrointestinal absorptionof the ACC is significantly higher than that of CCC (area under curve,AUC, values are higher by 22.5% and 20%, respectively, p<0.05), whilethe time required to reach the maximal concentration (T_(max)) did notdiffer between groups (Table 1).

The retention values presented in FIG. 1 suggest that rats that receivedCCC-containing capsules retained 48.5±1.3% of the received dose. On theother hand, rats that received ACC capsules retained 61.4±2.0% and60.6±2.1% of the received dose, respectively. This corresponds to asignificant increase in retention of 26.6%, as compared to the retentionby the CCC-treated group (p<0.05).

Experimental Details Animals

All animals were treated according to the Israel Animal Welfare Actunder the supervision of the Ben-Gurion University Animal Care and Useprogram. Fifty-one two month-old male Wistar rats (Harlan-Teklad,Jerusalem, Israel), weighing 240±15 g, were randomly housed in 12stainless steel cages in an environmentally-controlled room (23° C.temperature, 12:12 h light:dark cycle). The rats were fed laboratory ratchow pellets adequate in nutrients ad libitum (Koffolk, Petah-Tikva,Israel) and had free access to water for 48 h. Four days before thebeginning of the experiment, the regular diet was replaced with a lowcalcium diet containing 0.24±0.05% calcium (0.675±0.05% phosphate),specially prepared by mixing two food types containing, respectively,0.01% calcium (0.3% phosphate; Harlan-Teklad) and 1% calcium (0.8%phosphate; Koffolk). The two food types were separately ground in a millto yield two powders which were dry mixed in a ratio of 4:1,respectively, until fine homogenization. The homogenized powder wasextruded to form new food pellets.

Seventeen hours prior to capsule administration, the rats were weighedand blood samples were taken (baseline). The rats were then placed intoindividual metabolic cages and deprived of food and water until 3 hpost-capsule administration. Three and 24 h post-dosing, ˜10 g of lowcalcium food pellets (0.01% calcium; Harlan-Teklad) were given to eachrat. Distilled water was allowed ad libitum starting three hpost-capsule administration.

Gelatin Capsule Administration to Animals

Each rat was lightly sedated for 30 seconds with isoflurane (Minrad)diluted 1:4 (v:v) with propylene glycol (BioLab). A single capsulecontaining a specific calcium carbonate preparation (i.e. CCC, ACC, orACC-C; n=17 for each group) was administered intragastrically to each ofthe experimental rats using a stainless steel rat administration syringe(Harvard Apparatus).

Animals Blood Sampling and Chemical Analysis

Blood samples of 120-150 μl were taken from each rat's tail vein 17 hprior to capsule administration (time 0) and 2, 3, 6, 10, 24 and 34 hpost-administration. The blood samples were immediately centrifuged for10 minutes at 3000 g using a tabletop centrifuge (Hettich Zentrifugen,Bach, Switzerland). Duplicate samples (30 μl) of the supernatant serumwere transferred into plastic vials containing scintillation liquid(Zinsser Analytic, Berkshire, UK) and radioactivity was measured using aliquid scintillation counter (Tri-Carb 2100TR, PerkinElmer, Boston,Mass.). Plasma radioactivity from the given dose were normalizedaccording to the measured radioactivity and specific radioactive dosethat each rat received [(serum cpm×100)/(total cpm×sample volume)].

Animal Feces and Urine Sampling and Analysis)

Feces and urine were collected during the 17 h starvation phase duringthe acclimation period (baseline) and during the entire 34 h of theexperiment to evaluate calcium. Samples of urine (500 μL) weretransferred into plastic vials filled with scintillation liquid. Feceswere dried overnight at 70° C. in an oven. The samples were ground in amortar until fine homogenization. Feces samples (200 mg) were placedinto 5 ml of 1 N NaOH solution (Gadot, Netanya, Israel), incubated for 3hours at 80° C. and centrifuged for 10 minutes at 3,600 g. Duplicatesamples of the supernatant (30 μL) were transferred into plastic vialscontaining scintillation liquid. Radioactivity of the urine and fecessamples was measured using a liquid scintillation counter. Retentionvalues were calculated by subtracting the radioactivity measured in thefeces and urine from the given dose [(intake−feces and urineexcretion)/(intake×100%)].

Pharmacokinetic Calculations of Animal Samples

Non-compartmental analysis of an individual rat's calcium concentrationversus time data was performed using WinNonLin 5.2 software (Pharsight,Mountain View, Calif.).

Statistical Analysis

One-way analysis of variance (ANOVA) was performed on retention values,pharmacokinetic results and solubility results using the Statistica 6.1software (StaSoft, Tulsa, Okla.).

Paired t-test and student's t-test were performed using Prism 5software.

A p value<0.05 was deemed significant.

The results of the above trial confirmed that stable ACC has highergastrointestinal absorption than CCC in an animal model.

Trial 2: Calcium Gastrointestinal Absorption in Hypoparathyroidism

The experiment aiming to evaluate gastrointestinal bioavailability ofthe calcium source comprising stable Amorphous Calcium Carbonate (ACC)in the treatment of calcium malabsorption is conducted. A randomized,two phase, adaptive then crossover open-label, study is performed,comparing amorphous calcium carbonate (ACC) supplement to commerciallyavailable crystalline calcium supplements (CCS) in the management ofprimary hypoparathyroidism.

The primary objective of the first phase of the trial is a proof ofconcept that treatment with smaller doses of elemental calcium from ACCcompared to crystalline calcium supplement (CCS) can maintain targetserum calcium (corrected for albumin) values (7.0-10.0 mg/dL). Thesecondary objective of the first stage is to evaluate the sufficient ACCdose. The further secondary objective is to determine the effect of foodon ACC absorption.

The primary objective of the second phase of the trial is testing thehypothesis that treatment with smaller doses of elemental calcium fromACC compared to CCS can maintain target serum calcium (corrected foralbumin) values (7.0-10.0 mg/dL). The secondary objective of the firststage is testing the hypothesis that treatment with smaller doses ofelemental calcium from ACC compared to CCS does not cause an increase inhypercalciuria in subjects with hypoparathyroidism. The furthersecondary objective is testing the hypothesis that treatment withsmaller doses of elemental calcium from ACC compared to CCS reduces theside effects related with high calcium consumption.

Selection of Study Population

The study population includes twenty (20) subjects with primaryhypoparathyroidism, 10 subjects for each phase.

Investigational Product

The stable amorphous calcium carbonate used in the study is a syntheticACC stabilized by low concentrations of phosphoserine and citrate (lessthan 0.5% in the final product), provided by Amorphical Ltd.Phosphorylated serine and the organic citric acid are non-toxic,naturally abundant and even consumed as a standalone dietary supplementswith no reported adverse effects when taken orally.

Table 2 summarizes the ACC chemical analysis, assessed according to theU.S. Pharmacopeia parameters of calcium carbonate, using InductivelyCoupled Plasma Atomic Emission (ICP-AE), Ultraviolet (UV) Spectroscopy,Loss on Ignition (LOI) and flame photometer. Unless otherwise specified,the accuracy of the measured values is ±10%.

TABLE 2 Chemical analysis of Amorphical ACC. Analysis USP requirementsACC analysis result Loss On Ignition (LOI) N/A 14.8% Ethanol residuesN/A 0.034% Acid Insoluble Less than 0.2% Less than 0.0002% Calcium N/A32.5% Chlorides N/A 1.36% Sodium N/A 1.85% Phosphorus N/A 0.143% SulfurN/A <0.1% Iron Less than 1,000 ppm 9.5 ppm Alkali metals* Less than10,000 ppm 475 ppm Barium** Pass flame test 20 ppm Mercury Less than 0.5ppm Less than 1 ppm*** Fluorides Less than 0.005% 0.001% Lead Less than3 ppm Less than 5 ppm*** Heavy metals Less than 0.002% Trace (Less than0.002%) Arsenic (total) Less than 3 ppm Less than 3 ppm Crystalline Lessthan 5% Less than 1% Calcium Carbonate *Not including sodium. **Cannotbe performed according to the USP (was performed in Inductive CoupledPlasma (ICP)). ***Interferences in the ICP measurements prevented lowerconcentration analyses.

Control Product—Standard of Care

Treatment of patients with hypoparathyroidism involves correcting thehypocalcemia by administering calcium and vitamin D (Cusano et al, 2012;Fong & Kahn, 2012). Oral supplementation is started with elementalcalcium (1-2 g 3 times daily) and calcitriol (0.25-1 μg 2 or 3 timesdaily) for immediate management of postsurgical hypoparathyroidism. Inpatients at risk of severe and/or prolonged hypocalcemia, elementalcalcium is started at a dosage of 2 g 3 times daily and calcitriol at adosage of 0.5 μg 3 times daily. There are several types of calciumsupplementation. It is available with or without a prescription. All ofthem moderates nerve and muscle performance and facilitates normalcardiac function (Straub, 2007):

-   -   Calcium Carbonate (Tums extra strength, Cal-plus, Caltrate,        Os-Cal 500)—Amongst all available calcium supplements'        formulations, calcium carbonate is one of the most concentrated        calcium supplements with 40% elemental calcium. Many        commercially available preparations exist. Total daily dose of        elemental calcium needs to be titrated to minimize the daily        dose of vitamin D and to keep patients asymptomatic. Ionized        calcium is absorbed best in an acidic environment; 400 mg        elemental calcium equals 1 g calcium carbonate.    -   Calcium Citrate (Citracal, Cal-Cytrate 250)—210 mg of elemental        calcium equals 1 g calcium citrate.    -   Calcium Gluconate (Kalcinate)—Available for IV use. Infuse        slowly over 5-10 min; 10 mL calcium gluconate contains        approximately 90 mg elemental calcium; 1000 mg of calcium        gluconate equals 90 mg elemental calcium.

In the present study, patients in the control group continue to consumetheir regular calcium supplements and thus are treated withstandard-of-care.

Dosage and Administration Phase 1

Eligible subjects receive ACC tablets. Each dose of the studyinvestigational product consists of 50 or 200 mg of elemental calciumfrom ACC in each tablet.

Phase II

Eligible subjects receive ACC tablets in a crossover study design. Thecontrol arm is treated with a standard of care (by taking their regularcalcium supplementation). Each dose of the study investigational productconsists of 50 or 200 mg of elemental calcium from ACC in each tablet.

TABLE 3 Dose of Study Treatment Group Treatment INVESTIGATIONAL Tablets,each containing 50 or 200 mg of PRODUCT elemental calcium from ACC, fororal use. CONTROL PRODUCT Standard of care.

The mechanism of action and the effect of ACC are yet to be revealed.Therefore, selection of the dosage and the favorable absorptionconditions (fed/fasted) is based on the results of phase I of the study.While the final dosage has to be determined specifically for everypatient, it is expected that it will be possible to determine aconversion factor between ACC and CCS that will allow easy conversionbetween both formulas.

The calcium dosage in the control arm is determined according to theknown medical history and medication routine of each and every patient.

The tablets administration is performed 3 times a day throughout thetreatment period by the subject. IPs administration is documented in theCase Report Forms (CRF) in each visit to the Clinical Research Center(CRC) IP Administration Records. Packs with unused tablets are returnedto the CRC unit in visits days, counted and documented in the CRFs.

Allocation of Subjects to Treatment

Subjects are assigned to one of the treatment groups randomly accordingto a randomization list. Randomization is performed using blockrandomization.

Blinding

This is an open-label study. The subjects, the investigators and anypersonnel involved in subjects' assessment, monitoring, analysis anddata management are not blinded to the subject formulation assignment.The Sponsor is responsible for preparing, dispensing and labeling theinvestigational product IP.

Study Design

The study consists of two phases:

Phase I

Ten (10) subjects previously diagnosed and chronically treated forprimary hypoparathyroidism are enrolled. The daily CCS intake isgradually replaced by reduced amount of elemental calcium from ACC. Five(5) subjects consume the ACC before having a meal and the other five (5)subjects consume the ACC after having a meal. The safety and theefficacy of the treatment are closely monitored throughout this phase.

The absorption of ACC is evaluated using weekly serum calcium correctedfor albumin (CA) value tests. Excretion of calcium in urine is tested atscreening and at the end of phase I.

Phase II—Ten (10) new subjects previously diagnosed and chronicallytreated for primary hypoparathyroidism are enrolled.

The subjects are randomly assigned to one of the following treatmentsfor 6 weeks:

-   -   1. Standard of care—The same elemental calcium formulation and        dosage that was used routinely prior to the study.    -   2. ACC—The established dosage of elemental calcium from ACC        (based on the conversion factor and the fed/fasted conditions        found in phase I of the study).

The two formulations are administered with the regular daily dosage ofvitamin D (1-alfa D3).

At the end of the treatment, each group receives the alternativeformulation for another 6 weeks.

The superior absorption of ACC is evaluated using weekly blood tests tocalculate serum CA values. Excretion of calcium in urine is tested atscreening and in the end of each treatment.

The following is a detailed description of the study procedures:

Eligible subjects are treated as follows:

Phase I

I-Day−21 (+/−17) Screening: Subjects with a diagnosed primaryhypoparathyroidism (see section 4.1 for definitions), and who aretreated with calcium and vitamin D supplementation at least 1 year priorto the beginning of the study and are without major renal or hepaticdisease, are invited to the CRC. At the clinic, subjects areinterviewed, their medical history and their current medication aredocumented and they sign an informed consent form (ICF). Subjects arereferred to perform blood tests for serum calcium, P, creatinine andalbumin levels. Calculation of albumin corrected calcium (CA) isperformed. Subjects are instructed to perform 24 hour urine collectionfor Ca, P and creatinine. Subjects are asked to fill out a food andmedication diary for 3 consecutive days to evaluate their daily dietarycalcium intake. Women of childbearing age undergo a urine pregnancytest. Eligible subjects, complying with all inclusion criteria andhaving none of the exclusion criteria are enrolled to the study.

Subjects are informed by phone or on site whether they are eligible toenter the study.

I-Day 0: Eligible subjects arrive at the CRC where they are asked aboutany changes in their medical condition since their last visit. Bloodtests are performed to define serum calcium, P and albumin baselinevalues. Calculation CA at baseline is performed.

Subjects receive a pack of ACC tablets, each tablet containing 50 or 200mg elemental calcium (according to the daily total amount of calciumsupplementation, 14 day supply+5 spare tablets). The replacement of CCSwith ACC is calculated according to the following formula:

NTDC=ITDC−[0.1×ITDC (mg CCS)]+[0.05×ITDC (mg ACC)]

*ITDC—Initial total daily calcium intake (mg)**NTDC—New total daily calcium intake (mg)

10% (in mg) out of the initial total daily intake of elemental calciumis replaced by 5% of elemental calcium from ACC (in mg, calculated outof the initial total daily intake). The daily intake of vitamin Dremains the same.

The calculation for the number of tablets per day is performedspecifically for each subject (according to the daily dosage of calciumsupplementation) by the doctor.

Subjects are instructed to take the amount of ACC tablets a day, inaccordance with their individual calculated NTDC:

-   -   1. Five subjects are instructed to take tablets in the morning        after a meal, in midday after a meal and in the evening after a        meal.    -   2. Five subjects are instructed to take tablets in the morning        before having a meal, in midday before having a meal and in the        evening before having a meal.

Subjects are instructed to continue their routine medicationsconsumption during the trial.

I-Day 3 (±1): Subjects arrive at the CRC and their serum calcium, P andalbumin levels are tested. Calculation of CA is performed to excludehypocalcemia (Ca<7.0 mg/dL). If CA values are within the desired targetrange (7.0-10.0 mg/dl), subjects continue to take the calcium doses thatwere instructed on I-day 0. If CA values are below 7.0 mg/dl or above10.0 mg/dl, changes to the calcium intake are made, according to thedoctor's decision. I-Day 7 (±1): Subjects arrive at the CRC and theirserum calcium, P and albumin levels are tested. Calculation of CA isperformed:

a. Conversion Factor 0.5:

If CA=baseline, then 20% (in mg, calculated out of the initial totaldaily intake) of elemental calcium is replaced by 10% of elementalcalcium from ACC (in mg, calculated out of the initial total dailyintake).

NTDC=ITDC−[0.2×ITDC (mg CCS)]+[0.1×ITDC (mg ACC)]

b. Conversion Factor 0.75:

If CA<baseline, then 10% (in mg, calculated out of the initial totaldaily intake) of elemental calcium is replaced by 7.5% of elementalcalcium from ACC (in mg, calculated out of the initial total dailyintake).

NTDC=ITDC−[0.1×ITDC (mg CCS)]+[0.075×ITDC (mg ACC)]

CA<7.00 mg/dl enforces end of treatment.

c. Conversion Factor 0.25:

If CA>baseline, then 10% (in mg, calculated out of the initial totaldaily intake) of elemental calcium is replaced by 2.5% of elementalcalcium from ACC (in mg, calculated out of the initial total dailyintake).

NTDC=ITDC−[0.1×ITDC (mg CCS)]+[0.025×ITDC (mg ACC)]

Subjects are asked about any side effects or AEs that may have occurredand changes in concomitant medications since their last visit. Subjectsare asked about symptoms and signs related with a change in serumcalcium levels (tetany, facial grimacing, paresthesias, muscle aches,arrhythmia, depression). Subjects receive instructions regarding the newdoses of ACC and are reminded to take the amount of tablets a day inaccordance with their individual calculated NTDC), in the morning, inmidday and in the evening, before or after a meal (based on theirinitial assignment). Subjects are reminded to continue their routinemedications consumption during the trial.

I-Day 10 (±1): Subjects arrive at the CRC and their serum calcium, P andalbumin levels are tested. Calculation of CA is performed to excludehypocalcemia (Ca<7.0 mg/dL). If CA levels are within the desired targetrange (7.0-10.0 mg/dl), subjects continue to take the calcium doses thatwere instructed on I-day 7. If CA levels are below 7.0 mg/dl or above10.0 mg/dl, changes to the calcium intake are be made, according to thedoctor's decision.

I-Day 14 (±1): Subjects arrive at the CRC and their serum calcium, P andalbumin levels are tested. Calculation of CA is performed to excludehypocalcemia (Ca<7.0 mg/dL). If none of the conversion formulas (a-c,I-Day 7) resulted in serum calcium values within the desired targetrange (7.0-10.0 mg/dL), the study is terminated.

Subjects are asked about any side effects or AEs that may have occurredand changes in concomitant medications since their last visit. Subjectsare asked about symptoms and signs related with a change in serumcalcium levels (tetany, facial grimacing, paresthesias, muscle aches,arrhythmia, depression).

50% of the initial daily supplementation of CCS is replaced by ACC basedon the conversion factor found in I-day 7 (formulas a-c):

-   -   a. Conversion factor 0.5: 50% (in mg, calculated out of the        initial total daily intake) of elemental calcium is replaced by        25% of elemental calcium from ACC (in mg, calculated out of the        initial total daily intake).

NTDC=ITDC−[0.5×ITDC (mg CCS)]+[0.25×ITDC (mg ACC)]

-   -   b. Conversion factor 0.75: 50% (in mg, calculated out of the        initial total daily intake) of elemental calcium is replaced by        37.5% of elemental calcium from ACC (in mg, calculated out of        the initial total daily intake).

NTDC=ITDC−[0.5×ITDC (mg CCS)]+[0.375×ITDC (mg ACC)]

-   -   c. Conversion factor 0.25: 50% (in mg, calculated out of the        initial total daily intake) of elemental calcium is replaced by        12.5% of elemental calcium from ACC (in mg, calculated out of        the initial total daily intake).

NTDC=ITDC−[0.5×ITDC (mg CCS)]+[0.125×ITDC (mg ACC)]

Subjects receive a pack of ACC tablets, each tablet containing 50 or 200mg elemental calcium (according to their individual calculated NTDC, 14day supply+5 spare tablets). Subjects receive instructions regarding thenew doses of ACC and are reminded to take the amount of tablets a day inaccordance their individual calculated NTDC, in the morning, in themidday and in the evening, before or after a meal (based on theirinitial assignment). Subjects are reminded to continue their routinemedications consumption during the trial.

I-Day 21 (±1): Subjects arrive at the CRC and their serum calcium, P andalbumin levels are tested. Calculation of CA are performed to excludehypocalcemia (Ca<7.0 mg/dL). Subjects are asked about any side effectsor AEs that may have occurred and changes in concomitant medicationssince their last visit. Subjects are asked about symptoms and signsrelated with a change in serum calcium levels (tetany, facial grimacing,paresthesias, muscle aches, arrhythmia, depression). If CA levels arebelow 7.0 mg/dl, or above 10.0 mg/dl, subject is excluded from the study(based on the doctor's decision). If CA levels are within the desiredtarget range (7.0-10.0 mg/dL), a complete replacement of the dailysupplementation of CCS with ACC is performed, based on the conversionfactor found in I-day 7 (formulas a-c):

-   -   a. Conversion factor 0.5: 100% (in mg) of the elemental calcium        initial total daily intake is replaced by 50% of elemental        calcium from ACC (in mg, calculated out of the initial total        daily intake).

NTDC=ITDC−[ITDC (mg CCS)]+[0.5×ITDC (mg ACC)]

-   -   b. Conversion factor 0.75: 100% (in mg) of the elemental calcium        initial total daily intake is replaced by 75% of elemental        calcium from ACC (in mg, calculated out of the initial total        daily intake).

NTDC=ITDC−[ITDC (mg CCS)]+[0.75×ITDC (mg ACC)]

-   -   c. Conversion factor 0.25: 100% (in mg) of the elemental calcium        initial total daily intake is replaced by 25% of elemental        calcium from ACC (in mg, calculated out of the initial total        daily intake).

NTDC=ITDC−[ITDC (mg CCS)]+[0.25×ITDC (mg ACC)]

Subjects receive instructions regarding the new doses of ACC and arereminded to take the amount of tablets a day in accordance theirindividual calculated NTDC, in the morning, in the midday and in theevening, before or after a meal (based on their initial assignment).Subjects are reminded to continue their routine medications consumptionduring the trial.

Subjects receive a container to perform 24 hour urine collection priorto their next scheduled visit.

I-Day 27 (±1) by phone: Subjects are reminded to perform 24 hour urinecollection.

I-Day 28 (±1)—Termination of phase I: Subjects arrive at the CRC andtheir serum calcium, P and albumin levels are tested. Calculation of CAis performed. Subjects provide the container of 24 hour urine collectionfor Ca, P and creatinine to test calciuria. Subjects are asked about anyside effects or AEs that may have occurred and changes in concomitantmedications since their last visit. Subjects are asked about symptomsand signs related with a change in serum calcium levels (tetany, facialgrimacing, paresthesias, muscle aches, arrhythmia, and depression).

Phase I results are examined before deciding whether or not to embark onthe crossover portion of the study planned for Phase II. Phase I data issummarized, showing for each subject, by arm and overall therelationship between CA levels by amount of ACC replacement of CCSreceived.

Phase II

Ten (10) new subjects previously diagnosed and chronically treated forprimary hypoparathyroidism are enrolled.

The subjects are randomly assigned to one of the following treatmentsfor 6 weeks:

-   -   1. Standard of care (CCS)—The same elemental calcium dosage that        was used routinely prior to the study.    -   2. ACC—The established dosage of elemental calcium from ACC        (based on the conversion factor and the fed/fasted conditions        found in stage I of the study).

The two formulations are administered with the regular daily dosage ofvitamin D (1-alfa D3).

At the end of the treatment, each group receives the alternativeformulation for another 6 weeks.

The superior absorption of ACC is evaluated using weekly blood tests tocalculate serum CA values. Excretion of calcium in urine is tested atscreening and in the end of each treatment.

II-Day−21 (+/−17) Screening: Subjects with a diagnosed primaryhypoparathyroidism, and who had been receiving calcium and vitamin Dsupplementation at least 1 year prior to the beginning of the study andare without major renal or hepatic disease, are invited to the CRC. Atthe clinic, subjects are interviewed, their medical history and theircurrent medication are documented and they sign an informed consent form(ICF). Subjects are referred to perform blood tests for Calcium, P andalbumin levels. Calculation of CA is performed. Subjects are instructedto perform 24 hour urine collection for Ca, P and creatinine levels.Subjects are asked to fill out a food and medication diary for 3consecutive days to evaluate their daily dietary calcium intake. Womenof childbearing age undergo a urine pregnancy test. Eligible subjects,complying with all inclusion criteria and having none of the exclusioncriteria are enrolled to the study.

Subjects are informed by phone or on site whether they are eligible toenter the study.

II-Day 0: Eligible subjects arrive at the CRC where they are asked aboutany changes in their medical condition since their last visit. Bloodtests are performed to define serum calcium, P and albumin baselinevalues. CA values are calculated at baseline.

Subjects are randomly assigned to one of the following treatments:

-   -   1. Standard-of-care (CCS)—The same elemental calcium dosage that        was used routinely prior to the study.    -   2. ACC—The established dosage of elemental calcium from ACC        (based on the conversion factor and the fed/fasted conditions        found in stage I of the study).

Subjects that were assigned to the ACC treatment arm, receive acontainer with ACC tablets, each tablet containing 200 mg elementalcalcium (35 day supply+5 spare tablet). Subjects assigned to the CCStreatment arm continue to take their routine calcium supplementation.

The exact dosage of calcium supplementation is determined for eachsubject according the known medical history.

Subjects are instructed to continue their routine medicationsconsumption during the trial.

II-Day 3 (±1): Subjects arrive at the CRC and their serum calcium, P andalbumin levels are tested. Calculation of CA is performed to excludehypocalcemia (Ca<7.0 mg/dL). If CA values are within the desired targetrange (7.0-10.0 mg/dl), subjects continue to take the calcium doses thatwere instructed on II-day 0. If CA levels are below 7.0 mg/dL or above10.0 mg/dL, changes to calcium intake are made, according to thedoctor's decision.

II-Day 7 (±1): Subjects arrive at the CRC and their serum calcium, P andalbumin levels are tested. Calculation of CA is performed. Subjects areasked about any side effects or AEs that may have occurred and changesin concomitant medications since their last visit. Subjects are askedabout symptoms and signs related with a change in serum calcium levels(tetany, facial grimacing, paresthesias, muscle aches, arrhythmia,depression). Adjustments of ACC or CCS intake are performed ifnecessary.

Subjects are reminded to take the calcium supplementation according totheir assignment instructions.

Subjects are reminded to continue their routine medications consumptionduring the trial.

II-Day 10 (±1): Subjects arrive at the CRC and their serum calcium, Pand albumin levels are tested. Calculation of CA is performed to excludehypocalcemia (Ca<7.0 mg/dL). If CA levels are within the desired targetrange (7.0-10.0 mg/dl), subjects continue to take the calcium doses thatwere instructed on II-day 7. If CA levels are below 7.0 mg/dL, or above10.0 mg/dL, changes to the calcium intake are made, according to thedoctor's decision.

II-Day 14 (±1): Subjects arrive at the CRC and their serum calcium, Pand albumin levels are tested. Calculation of CA is performed. Subjectsare asked about any side effects or AEs that may have occurred andchanges in concomitant medications since their last visit. Subjects areasked about symptoms and signs related with a change in serum calciumlevels (tetany, facial grimacing, paresthesias, muscle aches,arrhythmia, depression).

Subjects are reminded to take the calcium supplementation according totheir assignment instructions.

Subjects are reminded to continue their routine medications consumptionduring the trial.

II-Day 21 (±1): Subjects arrive at the CRC and their serum calcium, Pand albumin are tested.

Calculation of CA is performed. Subjects are asked about any sideeffects or AEs that may have occurred and changes in concomitantmedications since their last visit. Subjects are asked about symptomsand signs related with a change in serum calcium levels (tetany, facialgrimacing, paresthesias, muscle aches, arrhythmia, depression).

Subjects are reminded to take the calcium supplementation according tothe instructions.

Subjects are reminded to continue their routine medications consumptionduring the trial.

Subjects receive a container to perform 24 hour urine collection priorto their next scheduled visit.

II-Day 34 (±1) by phone: Subjects are reminded to perform 24 hour urinecollection.

II-Day 35 (±1): Subjects arrive to the CRC and their serum calcium, Pand albumin levels are tested. Calculation of CA is performed. Subjectsprovide the container with 24 hour urine collection for Ca, P andcreatinine to test calciuria. Subjects are asked about any side effectsor AEs that may have occurred and changes in concomitant medicationssince their last visit. Subjects are asked about symptoms and signsrelated with a change in serum calcium levels (tetany, facial grimacing,paresthesias, muscle aches, arrhythmia, depression).

Subjects previously assigned to the CCS treatment arm receive a pack ofACC tablets, each tablet containing 200 mg elemental calcium (35 daysupply+5 spare tablets). Subjects previously assigned to the ACCtreatment arm, are instructed to resume their regular CCSsupplementation.

The exact dosage of calcium supplementation for each subject isdetermined by the known medical history.

Subjects are reminded to continue their routine medications consumptionduring the trial.

II-Day 38 (±1): Subjects arrive at the CRC and their serum calcium, Pand albumin levels are tested. Calculation of CA is performed to excludehypocalcemia (Ca<7.0 mg/dL). If CA levels are within the desired targetrange (7.0-10.0 mg/dl), subjects continue to take the calcium doses thatwere instructed on II-day 35. If CA levels are below 7.0 mg/dl or above10.0 mg/dl, changes to the calcium intake are made, according to thedoctor's decision.

II-Day 42 (±1): Subjects arrive at the CRC and their serum calcium, Pand albumin levels are tested. Calculation of CA is performed. Subjectsare asked about any side effects or AEs that may have occurred andchanges in concomitant medications since their last visit. Subjects areasked about symptoms and signs related with a change in serum calciumlevels (tetany, facial grimacing, paresthesias, muscle aches,arrhythmia, depression). Adjustments of ACC or CCS intake are performedif necessary.

Subjects are reminded to take the calcium supplementation according totheir assignment instructions.

Subjects are reminded to continue their routine medications consumptionduring the trial.

II-Day 45 (±1): Subjects arrive at the CRC and their serum calcium, Pand albumin levels are tested. Calculation of CA is performed to excludehypocalcemia (Ca<7.0 mg/dL). If CA levels are within the desired targetrange (7.0-10.0 mg/dl), subjects continue to take the calcium doses thatwere instructed on II-day 42. If CA levels are below 7.0 mg/dl or above10.0 mg/dl, changes to the calcium intake are made, according to thedoctor's decision.

II-Day 49 (±1): Subjects arrive at the CRC and their serum calcium, Pand albumin levels are tested. Calculation of CA is performed. Subjectsare asked about any side effects or AEs that may have occurred andchanges in concomitant medications since their last visit. Subjects areasked about symptoms and signs related with a change in serum calciumlevels (tetany, facial grimacing, paresthesias, muscle aches,arrhythmia, depression).

Subjects are reminded to take the calcium supplementation according totheir assignment instructions.

Subjects are reminded to continue their routine medications consumptionduring the trial.

II-Day 56 (±1): Subjects arrive at the CRC and their serum calcium, Pand albumin levels are tested. Calculation of CA is performed. Subjectsare asked about any side effects or AEs that may have occurred andchanges in concomitant medications since their last visit. Subjects areasked about symptoms and signs related with a change in serum calciumlevels (tetany, facial grimacing, paresthesias, muscle aches,arrhythmia, depression).

Subjects are reminded to take the calcium supplementation according totheir assignment instructions.

Subjects are reminded to continue their routine medications consumptionduring the trial.

Subjects receive a container to perform 24 hour urine collection priorto their next scheduled visit.

II-Day 69 (±1) by phone: Subjects are reminded to perform 24 hour urinecollection.

II-Day 70 (±1)—Termination of phase II: Subjects arrive at the CRC andtheir serum calcium, P and albumin levels are tested. Calculation of CAis performed. Subjects provide the container with 24 hour urinecollection for Ca, P and creatinine to test calciuria. Subjects areasked about any side effects or AEs that may have occurred and changesin concomitant medications since their last visit. Subjects are askedabout symptoms and signs related with a change in serum calcium levels(tetany, facial grimacing, paresthesias, muscle aches, arrhythmia,depression).

An unscheduled visit may be performed at any time during the study atthe subject's request or as deemed necessary by the investigator. Thedate and reason for the unscheduled visit is recorded. All assessmentsare optional and to be conducted at the discretion of the investigator.

Early discontinuation visit occurs if a subject prematurely discontinuedstudy participation (for the reasons specified in Section 7.4) and issimilar to the Termination Visit (Day 28 in phase I, Day 70 in phaseII).

The study procedures are summarized in tables 4 and 5.

Phase I

TABLE 4 Phase I procedure schedule. Day Assessment/ (−21) *(−2) 0 3 7 1014 21 27 28 Evaluation (±21) (±2) (±1) (±1) (±1) (±1) (±1) (±1) (±1)(±1) CRC phone call X X CRC visit X X X X X X X X Serum CA test X X X XX X X X Serum Albumin test X X X X X X X X Serum P test X X X X X X X X24 h urine collection: X X Ca test 24 h urine collection: X X Creatininetest 24 h urine collection: X X P test Pregnancy test** X Food andmedication X diary Symptoms and signs X X X X related with abnormalserum Ca levels: questionnaire IP supply X X Adverse event Ongoing*Patients are informed about their eligibility to participate in thestudy **For women of childbearing age

Phase II

TABLE 5 Phase II procedure schedule. Day Assessment/ −21 *−2 0 3 7 10 1421 34 35 38 42 45 49 56 69 70 Evaluation ±21 ±2 ±1 ±1 ±1 ±1 ±1 ±1 ±1 ±1±1 ±1 ±1 ±1 ±1 ±1 ±1 CRC phone call X X X CRC visit X X X X X X X X X XX X X X Serum CA test X X X X X X X X X X X X X X Serum Albumin X X X XX X X X X X X X X X test Serum P test X X X X X X X X X X X X X X 24 hurine X X X collection: Ca test 24 h urine X X X collection: Creatininetest 24 h urine X X X collection: P test Pregnancy test** X Food and Xmedication diary Symptoms and X X X X X X X X signs related withabnormal serum Ca levels: questionnaire IP supply X X Adverse eventOngoing *Patients are informed about their eligibility to participate inthe study. **For women of childbearing age

Pre-Study (Screening) Procedures

The following screening assessments are performed at screening for eachsubject:

-   -   Sign an inform consent form    -   Subject Medical history    -   Subject current medication    -   Subject Interviews    -   Pregnancy test (for women of childbearing age)    -   Food and medication diary for 3 consecutive days    -   Compliance with inclusion/exclusion criteria

Pre-Dose Procedures

The following assessments are performed for each subject:

-   -   Subject blood test for calculation of serum CA value, P and        albumin    -   24 hours urine collection for calcium, P and creatinine

Study Tests Blood Tests

Blood tests are performed for each subject in each CRC visit from day 0and onward: Phase I: Day 0, 3, 7, 10, 14, 21, 28.

Phase II: Day 0, 3, 7, 10, 14, 21, 35, 38, 42, 45, 49, 56, 70.

24 Hour Urine Collection Tests

24 hour urine collection test is performed at:

Phase I: Termination of study (Day 28)

Phase II: Day 35 and termination of study (Day 70)

Symptoms and Signs Related with Hypocalcemia

A list of questions aimed to identify symptoms and signed related withhypocalcemia is asked during the following CRC visits:

Phase I: Day 7, 14, 21, 28

Phase II: Day 7, 14, 21, 35, 42, 49, 56, 70

Study Measures Blood Tests—Albumin Corrected Calcium (Ca) Calculation,Phosphorus and Albumin Serum Levels Calcium and Albumin:

The initial assessment of hypocalcemia is usually based on themeasurement of serum total calcium corrected for albumin concentration.Normal CA values range from 8.5 to 10.2 mg/dL. In subjects withhypoparathyroidism, the desired target CA values are 7.0-10.0 mg/dL.

The relationship between total serum calcium and albumin is defined bythe following rule: the serum total calcium concentration falls by 0.8mg/dL for every 1-g/dL fall in serum albumin concentration. This ruleassumes that normal albumin equals 4.0 g/dL and normal calcium is 10.0mg/dL.

Calcium (corrected,mmol/L)=Calcium (measured,mmol/L)+{(40−albumin(g/L))×0.02}  Calculation:

Phosphorus (P):

The serum phosphorus test measures the amount of phosphate in the blood.Normal values range from 2.4-4.1 mg/dL.

24 Hour Urine Collection Tests

For a 24-hour urine collection, all of the urine over a 24-hour timeperiod must be collected. The urine sample must include the last urine,24 hours after starting the collection.

Calcium:

Test results may reflect dietary intake:

TABLE 6 Calcium in urine Low amount of 50-150 milligrams (mg)/24-1.25-3.75 millimoles calcium in diet: hour sample (mmol) per day Averageamount 100-250 mg/24-hour sample 2.5-7.5 mmol per day of calcium indiet: High amount of 250-300 mg/24-hour sample 6.2-7.5 mmol per daycalcium in diet:

Urine calcium level>300 mg/24 hours or >4 mg/kg of weight/24 hours isconsidered as hypercalciuria.

Creatinine:

A creatinine clearance test is done on a sample of urine collected over24 hours. It is used to determine glomerular filtration rate, whichhelps to measure how well the kidney functions.

TABLE 7 Creatinine in urine Creatinine Men (younger than 40 years):107-139 milliliters per clearance: minute (mL/min) or 1.8-2.3milliliters per second (mL/sec) Women (younger than 40 years): 87-107mL/min or 1.5-1.8 mL/sec Creatinine clearance values normally go downwith age (normal values go down by 6.5 mL/min for every 10 years pastthe age of 20). *The normal adult urine calcium/creatinine ratio is <220mg/g.

Phosphorus:

The phosphate urine test measures the amount of phosphate in a sample ofurine collected over 24 hours (24-hour urine test). Phosphate is acharged ion that contains the mineral phosphorus.

TABLE 8 Phosphate in urine Adults: 0.4-1.3 grams (g) per 24- 13-42millimoles (mmol) hour urine sample per day Calcium- and Less than 1.0 gper 24-hour Less than 32 mmol per day phosphate- urine sample restricteddiet:

Results of a test to measure phosphate in urine are seldom useful ontheir own. They should always be interpreted along with the results ofother tests. Calcium and phosphate levels are often measured at the sametime.

Assessment of Symptom and Signs Related with Hypocalcemia

Subjects are asked to answer questions for the presence of symptoms andsigns related with hypocalcemia (tetany, facial grimacing, paresthesias,muscle aches, arrhythmia, and depression).

Subjects' answers are documented in the CRF.

Safety of Treatment Calcium, P and Albumin Serum Levels

Normal CA values range from 8.5 to 10.2 mg/dL. In subjects withhypoparathyroidism, the desired target CA values are 7.0-10.0 mg/dL. Theinitial assessment of hypocalcemia is usually based on the measurementof serum total calcium corrected for albumin concentration, according tothe following formula: Calcium (corrected, mmol/L)=Calcium (measured,mmol/L)+{(40−albumin (g/L))×0.02}.

The serum phosphorus test measures the amount of phosphate in the blood.Normal values range from 2.4-4.1 mg/dL.

Urine Calcium, P and Creatinine Tests

Urine calcium level>300 mg/24 hours or >4 mg/kg of weight/24 hours isconsidered as hypercalciuria.

Normal Urine creatinine levels in men (younger than 40 years) are107-139 milliliters per minute (mL/min) or 1.8-2.3 milliliters persecond (mL/sec) and in women (younger than 40 years) are 87-107 mL/minor 1.5-1.8 mL/sec. Creatinine clearance values normally go down with age(normal values go down by 6.5 mL/min for every 10 years past the age of20). Urine calcium measurement is expressed in relation to creatinine.

A normal reference interval for the urine calcium (mg/dL): urinecreatinine (mg/dL) ratio is <0.14.

Normal phosphate levels are 0.4-1.3 grams (g) per 24-hour urine sample.

Complications

Symptoms and signs related with hypocalcemia (tetany, facial grimacing,paresthesias, muscle aches, arrhythmia, depression) are documents.

Statistical Analysis

This is a two-stage prospective study in which ACC is evaluated as areplacement for CCS in subjects with hypoparathyroidism. Efficacy isevaluated by the degree to which smaller doses of ACC replacing largerdoses of CCS are able to provide the supplemental calcium required byindividuals with hypoparathyroidism. Safety is evaluated by adverseevents (AEs) including those related to urinary calcium excretions.Statistical plan and analysis are carried out by TechnoSTAT Ltd.

Design Considerations

This is a two-phase study in which the first phase evaluates apredetermined conversion factor from CCS to ACC in subjects withhypoparathyroidism. During this phase progressively larger proportionsof CCS are replaced with ACC depending on subject response, hence theadaptive nature of this phase.

If the first phase of the trial demonstrates the safety of theconversion factor, the second phase is initiated. The second phasecomprises a crossover trial with newly recruited subjects; i.e. subjectswho did not participate in the first phase of the trial. Each of thesesubjects is block randomized in 1:1 ratio to CCS Control or ACCTreatment. In the second leg of the crossover each subject receives thealternative formulation which he or she did not receive in the firstleg.

The study duration per subject participating in phase I is 49 days fromscreening (day −21) until termination (day 28) and 91 days fromscreening (day −21) until termination (day 70) per subject participatingin phase II.

Visits to the CRC takes place on days 0 (baseline), 3, 7, 10, 14, 21 and28 (end-of-phase I) and on days 0 (baseline), 3, 7, 10, 14, 21, 35, 38,42, 45, 49 and 70 (end-of-phase II) for phase I and phase II,respectively.

Blood and urine tests and clinical questionnaire are performed duringsubjects' visits at the clinic.

Analysis Sets Safety Population

The safety population consists of all patients in both study stages forwhom ACC or CCC were administered as part of trial procedures.

Efficacy Population

The efficacy population consists of Phase II subjects with no majorentry violations likely to affect outcome, for whom there are baselineand at least some post-treatment data on serum CA levels. The degree towhich an entry violation is likely to affect outcome is determined by areviewer blind to outcome (i.e. blind review).

Treatment of missing values: only observed data is used; missing data isnot imputed.

Endpoints Safety

Overall incidence of product related adverse events.

All adverse events and serious adverse events are collected andreported.

Primary Efficacy

-   -   In Phase I: Change in elemental calcium doses from ACC achieving        normalization in serum CA and urine calcium levels    -   In Phase II: Serum CA and urine calcium levels

Secondary Efficacy

Secondary efficacy endpoints include:

-   -   Side effects associated with calcium levels    -   Symptoms related to hypocalcemia    -   Serum phosphorous levels

Data Analysis Overview

Safety and subject disposition analyses are conducted on the safetypopulation. Efficacy analyses are done on the efficacy population. PhaseI and II results are analysed and presented separately.

The data is summarized in tables listing the mean, standard deviationmedian, minimum, maximum and number of subjects for continuous data, orin tables listing count and percentage for categorical data whereappropriate. Tables are presented by study arm, over time and overall.In Phase I, study arms consist of ACC intake before or after meals. InPhase II, study arms consist of CCS or ACC.

All statistical analyses are performed and data appendixes are createdusing the SAS® system. The effects of noncompliance, dropouts, andpossible covariates such as Age and Gender are assessed to determine theimpact on the general applicability of results from this study.

Subject Disposition

Subject disposition is tabulated; the number of enrolled, exposed,prematurely terminated and completed subjects is summarized

A list of dropouts is prepared including reason for discontinuation, andtime of discontinuation.

Safety

The safety analyses are descriptive and narrative in nature, with SAE'sand AE's coded using MedDRA and tabulated by body system, preferredterm, study arm, severity and relation to procedure. Descriptivestatistics and shift analysis tables, indicating changes between“normal” and “abnormal” values, are provided as appropriate forlaboratory tests.

Primary Efficacy Analyses: Phase I

Phase I data is summarized numerically and graphically, showing for eachsubject, by arm and overall the relationship between calcium levels byamount of ACC replacement of CCS received. ACC and serum CA levels aredescribed over time for each individual, by arm and overall,numerically, by shift tables and graphically.

Primary Efficacy Analyses: Phase II

Serum CA levels are summarized both numerically and graphically by armover time and for differences between arms over time for parallel,paired time points (D0,D35; D3,D38; D7,D42; D10,D45; D14,D49; D21, D56).Differences in CA levels between arms at parallel time points arecompared by paired t-test or Wicoxon Sign-rank test depending on thedistribution of differences.

Secondary Analyses

Side effects associated with a change is serum calcium levels as well assymptoms related to hypocalcemia are described by arm in both Phase Iand Phase II. Serum phosphorous levels are described by arm over time inPhase I and Phase II, as well as by differences between arms at paralleltime points in Phase II. Shift tables are provided by arm for bothPhases.

Interim Analysis

There is no formally planned interim analysis within either of the twostudy phases. However, safety data, which includes serum CA andphosphorous levels, is monitored continuously. Phase I results areexamined before deciding whether or not to embark on the crossoverportion of the study planned for Phase II.

Data Management

Investigational data is recorded on paper CRFs. CRFs are filled forenrolled subjects only (not screening failures). Through his/hersignature on the CRF, the investigator certifies that the data collectedfor each patient are accurate, complete and legible.

All data generated in the current clinical investigation are managedaccording to TechnoSTAT standard procedures. Data and other studydocumentation are archived for a minimum of 6 years after the completionof the investigation.

Safety Definition of Adverse Events

An adverse event is any untoward medical sign (including an abnormallaboratory finding), symptom or disease temporally associated with theuse of the IPs whether or not considered as IP related. A new conditionor the worsening of a pre-existing condition is considered an AE. Allabnormal findings considered to be clinically significant must berecorded as adverse events.

The cases of acute medical situation (which are assessed within 48 hoursfrom the beginning of the study) that are considered significant by thePrincipal Investigator (such as pins and needles, numbness around themouth, cramps, anxiety, muscular contractions, seizures, stridor,psychosis, nausea, vomiting, loss of appetite, constipation, stomachpain, thirst, dry mouth and increased urination) are documented in theCase Report Form (CRF) and the subject is excluded from the experiment.

In all cases, the etiology of the AE should, as much as possible, beidentified and the Sponsor notified by recording in the CRF.

The relationship of the adverse event to the IP is defined as follows:

TABLE 9 The relationship of the adverse event to the IP Most probablyFollows a reasonable temporal sequence from study IP related:administration and cannot be reasonably explained by knowncharacteristics of the patient's clinical data Possibly Follows areasonable temporal sequence from study related: IP administration butcould have been produced by the patient's clinical state Probably notTemporal association is such that the study IP is not related: likely tohave had any reasonable association with the observed event. Notrelated: No relationship to study IP is perceived.

Serious adverse event (SAE) is any adverse event that:

-   -   Led to death.    -   Resulted in a life-threatening illness or injury.    -   Resulted in permanent impairment of a body structure or body        function    -   Required inpatient hospitalization or prolongation of existing        hospitalization.    -   Resulted in medical or surgical intervention to prevent        permanent impairment to body structure or body function    -   Led to fetal distress, fetal death, congenital abnormality or        birth defect.

Definitions of Adverse Event Intensity

The following definitions should be used by the investigating physicianto describe the intensity of adverse events.

The following are the only definitions which should be used to describeadverse event intensity. Only one severity definition should be used foreach adverse event (e.g. “mild-moderate” is not acceptable).

TABLE 10 Adverse Event intensity Intensity Definition Mild Adverseexperience which is easily tolerated Moderate Adverse experiencesufficiently discomforting to interfere with daily activity. SevereAdverse experience which prevents normal daily activities.

Abnormal Laboratory Values

A clinical laboratory abnormality is documented if any one of thefollowing conditions is met:

-   -   The laboratory abnormality is present in a repeated test    -   The abnormality is of a degree that requires active management        (e.g. change of dose, discontinuation of the treatment, more        frequent follow-up assessments, etc.).

Normal laboratory values are:

-   -   Serum calcium (albumin corrected): 7.0 to 10.0 mg/dL    -   Serum phosphorus: 2.4-4.1 mg/dL    -   Urine calcium: >300 mg/24 hours    -   Urine creatinine: 40 to 300 mg/dl    -   Urine phosphate: 0.4-1.3 grams (g)

Abnormal laboratory results that are not within the normal referencerange are recorded and treated.

Recording of Adverse Events

All of the following details should be recorded in the subject's CRF foreach adverse event:

-   -   Full description of adverse event.    -   Date and time of onset.    -   Date and time of resolution.    -   Severity of event, to be assessed by the investigating physician        in accordance with the definitions in Section 16.2.    -   Relationship to study IPs is assessed by the investigating        physician.    -   Action taken (if any).    -   Outcome and details of any further follow-up.

Anticipated Side Effects (Adverse Events)

The following is a list of ACC investigational product possible adverseevents. The list is based on prior experience from use of natural ACCand effects related to crystalline forms of calcium consumption:

-   -   Belching    -   Gas    -   Constipation    -   Nausea    -   Vomiting    -   Loss of appetite    -   Increased urination    -   Kidney damage    -   Confusion    -   Irregular heart rhythm.

Trial 3: Calcium Bone Absorption in Corticosteroids ConsumingIndividuals

The experiment aiming to evaluate the bioavailability of the calciumsource comprising stable Amorphous Calcium Carbonate (ACC) for bonemineralization in the subjects suffering from calcium malabsorptionassociated diseases, disorders and conditions is conducted. Amulticenter prospective, randomized, parallel, double-blind, activecontrolled study is performed, comparing the effect of amorphous calciumcarbonate (ACC) versus crystalline calcium carbonate (CCC) on bonemineral density of patients chronically treated with corticosteroids

The primary objective of the trial is to assess the efficacy oftreatment with calcium from ACC compared to CCC on preservation of bonemineral density in patients exposed to long-term treatment ofcorticosteroids. The secondary objective is to evaluate the effect ofACC compared to CCC on fractures' prevalence during the study period.The further secondary objective is to evaluate the effect of ACCcompared to CCC on bone metabolism biomarkers. The yet further objectiveof the trial is to evaluate the safety profile of ACC in thispopulation.

Selection of Study Population

The study population includes sixty (60) subjects taking continuouslong-term corticosteroid therapy, thirty (30) subjects in each treatmentgroup.

Description of the Investigational Product

The stable amorphous calcium carbonate used in the study is a syntheticACC stabilized by low concentrations of phosphoserine and citrate (lessthan 0.5% in the final product), provided by Amorphical Ltd., asdisclosed hereinabove.

Description of the Control Product

The control product contained in each capsule 500 mg of crystallinecalcium carbonate (200±5 mg elemental calcium) and 167 mg of sucrose.Elemental calcium level in the control product was equal to theelemental calcium level in the treatment group in order to evaluate theeffect of amorphous calcium carbonate compared to crystalline calciumcarbonate.

Dosage and Administration

Eligible subjects have randomly received one of the two treatments. Eachdose of the study supplement consisted of 667 mg of ACC or 500 mg CCC ineach capsule.

TABLE 11 Dose of Study Treatment Group Treatment Investigational Tabletsfor oral use containing 667 mg ACC (200 mg Product elemental calcium)Control Product Tablets for oral use containing 500 mg CCC (200 mgelemental calcium) and 167 mg of sucrose.

Selection of the dosage is based on the recommended daily intake ofelemental calcium for subjects of age 19-50 (1000 mg/day). The calciumdosage in the control is equaled to the elemental calcium levels in ACCproduct.

Allocation of Subjects to Treatment

Subjects are assigned to one of the treatment groups randomly accordingto a randomization list. Randomization to each of the two study arms isperformed using block randomization within center.

Blinding

The oral calcium treatments administered in the clinical trial areblinded. The subjects, the investigators and any personnel involved insubjects' assessment, monitoring, analysis and data management areblinded to the subject formulation assignment, except the Sponsor who isresponsible for preparing, dispensing and labeling the investigationalproduct. Blinded labels are affixed to the vials prior to dosing by theun-blinded Sponsor.

Randomization Procedures

The study is double blinded and therefore the CRC staff and the subjectremain blinded to the code assignments throughout the study. Prior toadministration, each subject is assigned with an individual number andis treated according to the predetermined computer generatedrandomization list. A computer-generated algorithm is used to assign thesubject into the treatment groups. The treatment compositions areprepared by the Sponsor and labels are affixed to the vials prior toshipping. The hospital pharmacists are instructed to dispense theproducts to the CRC according to the cohort assignment lists.

Study Design

Sixty (60) patients that are currently beginning long-term (≥6 months)glucocorticosteroid treatment are randomly assigned to one of two groups(N=30). Patients in the treatment group receive amorphous calciumcarbonate (ACC) and those in the active control group receivecrystalline calcium carbonate (CCC). Both formulations are supplementedwith vitamin D upon need, based on the doctor's decision. Safetyparameters are evaluated throughout the trial.

Patients admitting to the CRC due to various medical conditions areroutinely evaluated. Subjects that are beginning a long-term treatmentwith glucocorticosteroids due to their medical condition are consideredcandidates for the trial and are invited to the CRC for screening.

Screening (Day −7)—Subjects sign an informed consent form (ICF).Chemistry and hematology tests are performed: sodium, potassium,hemoglobin, sedimentation rate, leukocytes calcium (total,albumin-corrected), phosphate, alkaline phosphatase, creatinine, andalbumin are measured. Also, serum PTH, 25-hydroxyvitamin D, andthyroid-stimulating hormone (TSH) are tested. Urinary excretion ofcalcium and creatinine are measured. General health is examined bymedical history and physical examinations. Eligible subjects, complyingwith all inclusion criteria and none of the exclusion criteria areenrolled to the study.

Subjects are informed by phone or on site whether they are eligible toenter the study.

Visit 1 (Day 0)—Eligible subjects are invited to the CRC. DEXA scan isperformed at baseline. BMDs are measured for the lumbar spine (L2-4),femoral neck and whole body. Evidences for existing fractures aredocumented. Serum levels of albumin, calcium (Ca), phosphorus, andcreatinine (Cr), as well as urinary Ca and Cr are measured. Biochemicalmarkers of bone metabolism (serum osteocalcin (OCN), P1NP (totalprocollagen type 1 N-terminal propeptice), bone-type alkalinephosphatase (BAP) and urinary levels of type I collagen cross-linkedN-telopeptide (NTX)) are measured. Subjects randomly receive packs oftablets, each capsule containing 200 mg elemental calcium (210 tabletsfor 42 day supply+20 spare tablets, a total of 230 tablets) with one ofthe formulations (ACC or CCC). Subjects are instructed to take 5capsules a day for the first 6 weeks (days 0-42), 2 tablets in themorning and 3 tablets in the evening, after a meal. To minimize therisks for calcium related side effects, subjects who take calciumregularly, are instructed to discontinue their calcium supplementsintake throughout the trial. Subjects are advised to take vitamin D3supplementation based on the doctors' decision. Subjects are asked tokeep a daily diary to record their glucocorticoid use.

Visit 2 (Day 42±3)—Subjects are asked about any side effects or AEs thatmay have occurred. Subjects are asked about any fracture event thatoccurred since their last visit to the CRC. Subjects complete the TSQMquestionnaire with the representative of the CRC. Subjects receiveadditional packs of tablets, each tablets containing 200 mg elementalcalcium (a total of 210 tablets, 42 day supply+20 spare tablets) of thesame formulation (ACC or CCC) that has been received in the previousvisit. Subjects are reminded to take 5 tablets a day for the next 6weeks (days 42-84), 2 tablets in the morning and 3 tablets in theevening, after a meal. To minimize the risks for calcium related sideeffects, subjects who take calcium regularly, are reminded todiscontinue their calcium supplements intake throughout the trial.Subjects are advised to take vitamin D3 supplementation based on thedoctors' decision. Subjects are asked to keep a daily diary to recordtheir glucocorticoid use.

Visit 3—(Day 84±3)—DEXA scan is performed. BMDs are measured for thelumbar spine (L2-4), femoral neck and whole body. Pre-dose Serum levelsof albumin, calcium (Ca), phosphorus, and creatinine (Cr), as well asurinary Ca and Cr are measured. Biochemical markers of bone metabolismare measured as well. Subjects are asked about any side effects or AEsthat may have occurred. Subjects are asked about any fracture event thatoccurred since their last visit to the CRC. Subjects receive additionalpacks of tablets, each containing 200 mg elemental calcium (a total of210 capsules, 42 day supply+20 spare tablets) with the same formulationreceived in day 0. Subjects are instructed to take 5 tablets a day forthe next 6 weeks (days 84-126) 2 tablets in the morning and 3 tablets inthe evening, after a meal. To minimize the risks for calcium relatedside effects, subjects who take calcium regularly, are reminded todiscontinue their calcium supplements intake throughout the trial.Subjects are advised to take vitamin D3 supplementation based on thedoctors' decision. Subjects are asked to keep a daily diary to recordtheir glucocorticoid use.

Visit 4 (Day 126±3)—Subjects are asked about any side effects or AEsthat may have occurred. Subjects are asked about any fracture event thatoccurred since their last visit to the CRC. Subjects receive additionalpacks of tablets, each capsule containing 200 mg elemental calcium (atotal of 210 tablets, 42 day supply+12 spare tablets) of the sameformulation (ACC or CCC) that has been received in the previous visit.Subjects are reminded to take 5 tablets a day for the next 6 weeks (days126-168), 2 tablets in the morning and 3 tablets in the evening, after ameal. To minimize the risks for calcium related side effects, subjectswho take calcium regularly, are reminded to discontinue their calciumsupplements intake throughout the trial. Subjects are advised to takevitamin D3 supplementation based on the doctors' decision. Subjects areasked to keep a daily diary to record their glucocorticoid use and tobring it with them to the next visit.

Visit 5—(Day 168±1)—DEXA scan is performed. BMDs are measured for thelumbar spine (L2-4), femoral neck and whole body. Pre-dose serum levelsof albumin, calcium (Ca), phosphorus, and creatinine (Cr), as well asurinary Ca and Cr are measured. Biochemical markers of bone metabolismare measured as well. Subjects are asked about any fracture event thathas occurred since their last visit to the CRC. Subjects complete theTSQM questionnaire with the representative of the CRC. Subjects areasked about any side effects or AEs that may have occurred.

Outcome Measures

-   -   Preservation of Bone Mineral Density (BMD)—BMD is defined by        Dual Energy X-ray Absorptiometry (DEXA) scan. The DEXA results        are presented as Z-score (comparing the results of a specific        scan to other people at the same age, weight, ethnicity, and        gender). A Z-score of less than −1.5 raises concern of factors        other than aging as contributing to bone loss.    -   Fracture prevalence—fracture events are documented in each        visit.    -   Increase in bone formation markers    -   Reduction in bone resorption markers    -   Assessment of calcium side effects:

Safety Analysis

The safety analyses are descriptive and narrative in nature. The safetyendpoints are adverse events (AEs) and serious AEs (SAEs) whether or notrelated to study treatment. Also included are serum calcium levels andurine calcium and creatinine levels.

Efficacy Analysis

The primary efficacy endpoint is preservation of BMD, by Z-valuescalculated by DEXA scan. Hypotheses are tested by independent groupst-test on raw values if these are distributed approximately normal.Log-transform is used to normalize the data, if the data substantiallydeviates from normal. If after applying the latter the data stilldeviates from normal, the non-parametric Wilcoxon Rank-sum test is used.The trial is considered successful, if the Z-score is significantlysmaller in Treatment relative to Control.

The following are the study's secondary efficacy endpoints:

-   -   Reduction in fractures' frequency    -   TSQM (Treatment Satisfaction Questionnaire for Medication)        domains: A 14-item psychometrically robust and validated        questionnaire consisting of four scales: the effectiveness        scale, the side effects scale, the convenience and the global        satisfaction scale.

Trial 4: Calcium Gastrointestinal Absorption in Populations Susceptibleto the Development of Osteoporosis.

The objective of the present study was to evaluate the gastrointestinalabsorption of calcium from the ACC source in population susceptible tothe development of bone loss related disorders. The trial is a clinicalstudy that used the dual-stable calcium isotopes technique performed onpostmenopausal women.

Ten postmenopausal women (demographic data in Table 12), with no morethan 5 years from menopause were included in the study. All subjects,exhibiting BMI of 18-29, were apparently healthy and did not suffer fromany major medical illness or metabolic bone disorder. Exclusion criteriaincluded women who, on the basis of a food diary consumption, have anestimated daily calcium intake>1100 mg through combined diet (from bothsupplements and food), vitamin D deficiency exhibited by values<20 ng/mlin the serum, hypercalcemia, nephrolithiasis, inflammatory boweldisease, malabsorption, chronic diarrhea, use of antibiotics within thepast month and women suffering from digestive, hepatic, renal, orinflammatory diseases. Women who take oral steroids, anticonvulsants,bisphosphonates, estrogen compounds, calcitonin, or teriparatide withinthe past 6 months were also excluded from the study. Written informedconsent was obtained from each woman after approval of the protocol bythe Ethical Committee of Sourasky Medical Center, Tel-Aviv, Israel.

TABLE 12 Demographic data of 10 women that participated in the studyVariable Age, y   55 ± 3.2 Height, cm 159.3 ± 3.7  Weight, kg 66.8 ± 5.1BMI, kg/cm² 26.2 ± 1.9 PTH, pg/ml  30.3 ± 13.2 FSH, miu/ml  65.2 ± 29.325-OHD, ng/ml 29.9 ± 6.7 Results are means ± S.D

Fractional absorption analysis revealed that all 10 subjects showed asignificant elevation in calcium absorption when being administered ACCinstead of CCC.

Paired t-test analysis of the results obtained from 9 women thatreceived the capsules after breakfast showed a significant elevation offractional calcium absorption for ACC. The average increase in therelative absorption of calcium from ACC vs. CCC for each woman was 2.1fold (p<0.01; FIG. 2).

Overall as two separated treatment groups, there was a 1.9 elevation incalcium fractional absorption when women were administered with ACCcompared to CCC (p<0.05; FIG. 3).

One out of the ten women who received the capsules on an empty stomachafter an overnight fast presented a 4.6 fold elevation in calciumfractional absorption (FIG. 4).

Experimental Details Capsules and CaCl₂) Solution Preparation andLabeling

ACC (Amorphical Ltd.) was intrinsically labeled by dissolvingappropriate amount of it in 32% HCl together with ⁴⁴CaCO₃ (enriched to96.1%, CMR, Moscow, Russia), after which it was reprecipitated back toACC powder containing 192 mg elemental calcium (600 mg powder) labeledwith 15 mg ⁴⁴Ca per treatment. Overall, 3 batches of ⁴⁴ACC were preparedand sent to magnetic sector thermal ionization spectrometry (MAT 261;Finnigan, Bremen, Germany) for 44:42 molar ratio analysis, and tocalcium content measurement by atomic absorption (Analytical ResearchServices, Ben-Gurion University, Beersheva, Israel). Additional testsevaluating the amorphous content for each batch were performed: X-raydiffraction (XRD; The Nanotechnology Institution, Ben-GurionUniversity), quantitative polarized spectroscopy (QPS), polarizedoptical microscopy (POM) and dryness. CCC (Zifroni Chemicals SuppliersLtd., Rishon-Lezion, Israel) was intrinsically labeled by homogenizingappropriate amount of it with ⁴⁴CaCO₃ (enriched to 96%, CMR, Moscow,Russia) to reach CCC powder containing 192 mg elemental calcium (480 mgpowder) labeled with 15 mg ⁴⁴Ca per treatment. To match the volume inthe ACC capsules, sucrose (120 mg per treatment; FAGRON Gmbh & Co. KG,Rotterdam, The Netherlands) was added to each capsule. One batch of⁴⁴CCC was prepared and sent to magnetic sector thermal ionizationspectrometry (MAT 261; Finnigan, Bremen, Germany) for 44:42 molar ratioanalyses.

Isotopic i.v. ⁴²CaCl₂ solution preparation procedures were conducted byConcept for Pharmacy Ltd. Kfar-Saba, Israel under the laminar flow hoodto ensure sterility. The appropriate amount of ⁴²CaCO₃ (enriched to96.3%, CMR, Moscow, Russia) was dissolved in 37% HCl and mixed with0.45% NaCl. The pH was adjusted with 10N NaOH to 5.5. The solution wasforced through a 0.22μ sterilization filter into a sterile container.Individual doses (1.5 mg ⁴²Ca/dose) were transferred into sterile vialsfor later use. Aliquots were sent for sterility and pyrogenicity testing(Aminolab, Nes-Ziona, Israel) and for calcium content measurement byatomic absorption (Analytical Research Services, Ben-Gurion University,Beersheva, Israel) before use. All of i.v. solutions in the study weresterile and free of pyrogens.

Urine Collection and Analysis

Following each capsules administration, a timed 24-hour urine collectionwas performed for measurements of calcium, sodium, potassium, urea,creatinine and stable isotope analysis. Calcium absorption analyses wereperformed at Baylor College of Medicine Houston, Tex., USA, followingpreviously published methods (Yergey et al 1994). Ammonium oxalate wasused to precipitate calcium isotopes in the urine samples (Yergey et al1980). The amount of extracted calcium was then used for calcium isotoperatio measurements. This amount was determined using magnetic sectorthermal ionization spectrometry (MAT 261; Finnigan, Bremen, Germany).

Pharmacokinetic Calculations of Human Samples

Fractional absorption (α_(24h)) of dietary calcium was calculated asdescribed in (Yergey et al 1994). In brief, the ratio α_(24h) is anarithmetically valid representation of the ratio of the areas under theplasma disappearance curves for the two labels.

Statistical Analysis

The statistical analysis of variance was performed as described in trial1.

The results of the above trial confirmed that stable ACC has highergastrointestinal absorption than CCC both in human models, susceptibleto the development of bone related disorders. Such enhanced absorptionmay be beneficial in cases when higher calcium intake in required formaintaining bone mineral density, as the absorption of crystallinecalcium is limited and the excess of calcium may lead to adverseeffects.

Trial 5: Preventing or Delaying Bone Loss in Bone Mineral Density LossAssociated Disorders.

The objective of the present trial was to evaluate availability ofcalcium from the ACC source comprising various stabilizers (P-Ser,citric acid or sucrose), for bone mineralization process assessed byfemoral and vertebral bone mineral density (BMD) in osteoporosis ratmodel (ovariectomy; OVX).

In the study, four groups of rats were tested (3 ovariectomized and 1sham). The animals were treated according to Table 13.

Table 14 presents the mean food consumption and body weight of the ratsduring the trial. A significant weight increase was observed in thethree OVX groups compared to sham. This is despite the fact that foodconsumption was lower among all OVX groups compared to the sham group.

TABLE 13 Assignment of rats according to the treatment Group TreatmentSham Standard laboratory diet containing 1% elemental calcium (n = 10)(Altromin Spezialfutter GmbH & Co, #1320P Seelenkamp, Germany) adlibitum. OVX ACC Low calcium diet (TD.95027, Harlan Inc, Jerusalem,Israel) (P-Ser) enriched with 1% elemental calcium from synthetic ACC (n= 10) stabilized by 0.0275% phosphoserine (ACC_PS, SOP PSC002,Amorphical Ltd.) ad libitum. OVX ACC Low calcium diet (TD.95027, HarlanInc, Jerusalem, Israel) (CIT) enriched with 1% elemental calcium fromsynthetic ACC (n = 10) stabilized by 0.0275% citric acid (ACC_CIT, SOPPCA001, Amorphical Ltd.) ad libitum. OVX ACC Low calcium diet (TD.95027,Harlan Inc, Jerusalem, Israel) (SUC) enriched with 1% elemental calciumfrom synthetic ACC n = 10) stabilized by sucrose and sodium hydroxide(ACC SUC, SOP SUC001, Amorphical Ltd.) ad libitum. OVX = ovariectomy;ACC = amorphous calcium carbonate; PS = phosphoserine; CIT = citrate;SUC = sucrose

TABLE 14 Mean food consumption and total body weight of the rats at thebeginning and at the end of the experiment OVX Sham ACC_P-Ser ACC_CITACC_SUC Rat body weight (Day 0) 258 (16) 245 (15) 244 (11) 255 (14) Ratbody weight (Day 90) 299 (29) 337 (18) 341 (16) 357 (22) *Weight gain(%) 23.7^(a) 36.1^(b) 41.2^(b) 40.7^(b) *Mean food consumption  17.2(1.0)^(a)  14.0 (0.8)^(b)  14.5 (0.5)^(bc)  15.5 (0.7)^(c) Values wererecorded weekly. Weight gain represents the increased rate of weigh fromthe beginning to the end of the experiment. Values are represented asmean (SD). One-way ANOVA: p < 0.001. Letters represents Tukey's post hoccomparison tests.

As expected in this model, micro-CT scanning of the femur of the OVXgroups revealed a significant reduction in trabecular bone mineraldensity (Tb.BMD) and other morphometric parameters, i.e: bonevolume/tissue volume (BV/TV), trabecular number (Tb.N), trabecularthickness (Tb.Th); and a significant elevation in trabecular separation(Tb.Sp). In addition, the 3 OVX groups were significantly different fromthe sham animals in all femoral microarchitectural topology andorientation parameters, i.e.: trabecular bone factor (TBPf), structuralmodel index (SMI) and degree of anisotropy (DA) (Table 15).

Results of the 4-L vertebra micro-CT scan show that the sham group wassignificantly higher compared to all other OVX groups only in BMD, BV/TVand Tb.N parameters and significantly lower in TBPf and SMImicroarchitectural parameters (Table 15).

In contrast to the trabecular BMD, no differences between all groupswere observed in the cortical bone BMD (Ct. BMD) of both the femur andvertebra (Table 15).

In all parameters tested by the micro-CT, no difference was observedamong the ACC+P-Ser, ACC+CIT and the ACC+SUC groups in both femur andvertebra (Table 6), indicating that the compound that stabilizes ACC byitself has no effect on BMD or additional bone morphometric ormicroarchitectural parameters.

TABLE 15 Morphometric and microarchitectural topology & orientationparameters studied by micro-CT OVX Sham ACC + P-Ser ACC + CIT ACC + SUC(A) *Tb. BMD, g/cm³ 305 (43)^(a) 120 (26)^(b) 112 (20)^(b) 116 (27)^(b)Femur Ct. BMD, g/cm³ 775 (79)^(a) 771 (32)^(a) 761 (84)^(a) 743 (55)^(a)*Tb. BV/TV, % 55 (7.5)^(a) 22 (3.7)^(b) 21 (3.6)^(b) 22 (3.6)^(b) *Tb.N, mm⁻¹ 4.5 (0.3)^(a) 2.0 (0.3)^(b) 2.0 (0.3)^(b) 2.1 (0.3)^(b) *Tb. Sp,μm 145 (15)^(a) 583 (67)^(b) 621 (125)^(b) 613 (92)^(b) *Tb. Th, μm 121(1)^(a) 109 (6)^(b) 105 (6)^(b) 104 (6)^(b) *TBPf, mm⁻¹ −11.4 (4.0)^(a)5.20 (1.6)^(b) 5.99 (1.7)^(b) 4.60 (1.7)^(b) *SMI −0.84 (0.8)^(a) 1.40(0.1)^(b) 1.46 (0.2)^(b) 1.32 (0.2)^(b) *DA 1.46 (0.1)^(a) 1.64(0.2)^(b) 1.82 (0.2)^(b) 1.61 (0.2)^(b) (B) *Tb. BMD, g/cm³ 235 (41)^(a)107 (37)^(b) 124 (39)^(b) 130 (29)^(b) Vertebra Ct. BMD, g/cm³ 652(50)^(a) 687 (35)^(a) 663 (41)^(a) 669 (48)^(a) *Tb. BV/TV, % 42(6.6)^(a) 22 (5.1)^(b) 24 (5.4)^(b) 25 (5.3)^(b) *Tb. N, mm⁻¹ 3.6(0.5)^(a) 2.1 (0.4)^(b) 2.1 (0.9)^(b) 2.2 (0.4)^(b) Tb. Sp, μm 221(34)^(a) 335 (53)^(a) 297 (48)^(a) 333 (57)^(a) Tb. Th, μm 117 (5)^(a)105 (7)^(a) 107 (10)^(a) 111 (6)^(a) *TBPf, mm⁻¹ −0.99 (3.5)^(a) 7.04(2.5)^(b) 5.85 (2.8)^(b) 5.73 (1.7)^(b) *SMI 0.41 (0.5)^(a) 1.51(0.3)^(b) 1.56 (0.7)^(b) 1.40 (0.2)^(b) DA 2.03 (0.3)^(a) 2.64 (0.7)^(a)2.36 (0.4)^(a) 2.13 (0.4)^(a) (A) Distal femur metaphysis. (B)4^(th-)lumbar vertebra. Trabecular bone mineral density (Tb. BMD),cortical BMD (Ct. BMD), Trabecular bone volume/tissue volume (Tb.BV/TV), Trabecular number (Tb. N), trabecular separation (Tb. Sp),trabecular thickness (Tb.Th), Trabecular bone pattern factor (TBPf),structure model index (SMI), degree of anisotropy (DA). Valuescorrespond to means (±SD). One-way ANOVA: *p < 0.001. Letters representsTukey's post-hoc comparison tests.

Experimental Details Animals

Forty 16-17 weeks old female Sprague Dawley rats (Harlan Inc.,Jerusalem, Israel) with an average body weight of 247±14 g weregroup-housed in 14 cages (2-3 rats/cage) and acclimated under controlledroom conditions (21±2° C. and 12-hours dark-light cycle). During 7 daysacclimation, rats were fed with standard laboratory diet containing 1%elemental calcium (#2018SC Rat chow, Harlan Inc. Jerusalem, Israel) andde-ionized water ad-libitum.

Following the 7 days acclimation period, rats were anesthetized usingintraperitoneal (IP) injection of anesthetic solution (0.1 ml/100 g)containing Ketamine (Fort Dodge AH Ltd. Overland Park, USA) and Xylazine(EuroVet AH Ltd. Bladel, UK).

Rats were randomly assigned and operated according to their assignedfour groups: Sham (sham-Ovariectomized; fed with standard laboratorydiet containing 1% elemental calcium from CCC) and three other groupswere ovariectomized (OVX) using a bilateral dorsal approach according toLasota and Danowska-Klonowska (2004) Rocz Akad Med Bialymst 49 Suppl1:129-131, and divided to OVX-ACC+PS, OVX-ACC+CIT and OVX-ACC+SUC.Following operation, rats were provided a 7 days recovery period underthe same conditions as the acclimation period, standard laboratory dietad-libitum and de-ionized water containing 1.5 ml/400 ml Dipyrone(Vitamed Ltd, Binyamina, Israel), and 1 mg/kg Enrofloxacin (Buyer Ltd.Leverkusen, Germany).

Experimental Treatment

At the end of the recovery period the rats were group-housed (2rats/cage) according to the group assignment, under the same controlledroom conditions as described above. The assigned food pellets (describedin Table 13) and de-ionized water were provided ad-libitum during theentire treatment period (90 days). Food consumption and body weight wererecorded weekly (described in Table 14). For dynamic histomorphometricanalysis fluorochrom dye Calcein (20 mg/kg body mass; Sigma-Aldrich,Israel) was IP injected to all rats at days 76 and 86. On day 90, ratswere individually housed in metabolic cages and deprived from food andwater for 18 h. Urine was collected from each metabolic funnel and wasstored at −80° C. for future analysis of bone resorption biochemicalmarker at end of the experiment (Time 90) Immediately after urinesampling, serum samples were extracted for evaluation of formationbiochemical marker of day 90. All rats were sacrificed by CO₂. Rightfemurs and 4^(th) lumbar vertebras were dissected, wrapped with gazepads soaked with saline buffer and kept in −80° C. for measurement ofbone micro-CT scan. The 5^(th) lumbar vertebras were stored in the sameconditions as the right femurs for mechanical testing. Right tibias weredissected and placed immediately for 24 h at 4° C. in 3.7% formaldehydesolution (F1636, Sigma-Aldrich, Israel) for structural and dynamichistomorphometric measurements.

Micro-Computed Tomography (pCT) Scanning

Trabecular and cortical bone microarchitecture of right femurs and4^(th) lumbar vertebras were analyzed in a μCT scanner (Skyscan 1174,Kartuizersweg Kontich, Belgium). Calibration of the scanner for bonemineral density (BMD) was performed according to manufactureinstructions using a designated rat phantom rod with densities of 0.25and 0.75 g/cm³ (Skyscan, Kartuizersweg Kontich, Belgium).

Prior to scan analysis, femurs and 4th lumbar vertebras were removedfrom −80° C. and placed in a plastic (according to the manufactureguidance). The distal region of each femur was scanned for trabecularand cortical bone parameters at an isotropic resolution of 13.8 μm.Reconstruction was carried out employing a modified algorithm using theSkyscan Nrecon software (ver. 1.6.4, Skyscan). Volume of interest (VOI)was set to 0.8 mm below growth plate, and extended distally for 1.5 mm.Trabecular and cortical segments within the VOI were extracted bydepicting ellipsoid contours every ˜5 slices using the CT analysissoftware provided with the scanner (ver. 1.11 Skyscan). Trabecular andcortical BMD, bone volume (BV/TV, %), trabecular number (Tb.N),trabecular thickness (Tb.Th, μm), trabecular separation (Tb.Sp, μm)along with the micro-architectural parameters of trabecular bone patternfactor (TBPf) and structure model index (SMI) were calculatedautomatically by the CT analyzer software (ver. 1.11 Skyscan).

The 4^(th) lumbar vertebra from each rat was scanned in a 13.8 μmisotropic resolution and VOI was set to cover 1.25 mm thick crosssection from the center of the vertebral body for evaluation of thetrabecular and cortical parameters in the same procedure as describedabove for the femurs.

Trial 6: Increasing Bone Mineral Density in Metabolic Bone RelatedDisorders

The inventors have conducted a study aiming to evaluate the effects ofAmorphous Calcium Carbonate (ACC) on bone mineral density and bonefraction in metabolic bone disorders, and specifically osteoporosis aspresented in the ovariectomized (OVX) rat model wherein the ACC isadministered in combination with a standard medication for osteoporosis.In the present trial, five groups of rats were tested (4 OVX and 1sham). Following operation, all rats were left untreated (fed withstandard laboratory diet) to develop osteopenia for a period of 2months. At the end of the induction period, the animals were treatedwith special food mixtures in the form of pellets from two differentcalcium sources: amorphous calcium carbonate (ACC) or crystallinecalcium carbonate (CCC) (Table 16). Pellets from the two mixturescontained ˜1% elemental calcium as confirmed by atomic absorptionapparatus (Varian AA240, Palo Alto, Calif., USA) with no added vitaminD, except for the amounts found in the original food pellets (22 IUvitamin D/g). In addition, the animals were i.v injected (3 times/week)with either saline or 2 μg/kg alendronate (ALN) according to their groupassignment, as described in Table 16.

TABLE 16 Assignment of rats according to the treatment Group TreatmentSham Sham Low calcium diet (TDK95027, Harlan Inc.) containing 1%elemental calcium from CCC (Socal ® Precipitated Calcium Carbonate) adlibitum and subcutaneous injection of saline (0.9% NaCl, 100 μl/100 gb.w) 3 times\week. OVX CCC Low calcium diet (TDK95027, Harlan Inc.)containing 1% elemental calcium from CCC (Socal ® Precipitated CalciumCarbonate), ad libitum and subcutaneous injection of saline (0.9% NaCl,100 μl/100 g b.w) 3 times\week. ACC Low calcium diet (TDK95027, HarlanInc.) containing 1% elemental calcium from synthetic amorphous calciumcarbonate [ACC (1% PS & 2% Citrate) provided by Mr. Oren Meiron,Amorphical] ad libitum and subcutaneous injection of saline (0.9% NaCl,100 μl/100 g b.w) 3 times\week. CCC + ALN Low calcium diet (TDK95027,Harlan Inc.) containing 1% elemental calcium from CCC (Socal ®Precipitated Calcium Carbonate) ad libitum and subcutaneous injection ofalendronate (2 μg/kg as 100 μl/100 g b.w) 3 times\week. ACC + ALN Lowcalcium diet (TDK95027, Harlan Inc.) containing 1% elemental calciumfrom synthetic amorphous calcium carbonate [ACC (1% PS & 2% Citrate)provided by Mr. Oren Meiron, Amorphical] ad libitum, and subcutaneousinjection of alendronate (2 μg/kg as 100 μl/100 g b.w) 3 times\week. OVX= ovariectomy; CCC = crystalline calcium carbonate; ACC = amorphouscalcium carbonate; ALN = alendronate; PS = phosphoserine

Table 17 presents the mean food consumption and body weight of the ratsduring the trial. A significant weight increase was observed in all OVXrats compared to sham. This is despite the fact that food consumptionwas similar among all groups.

TABLE 17 Mean food consumption and total body weight of the rats OVXSham CCC ACC CCC + ALN ACC + ALN Rat body weight 258 (16) 244 (18) 240(21) 243 (14) 244 (16) (Day 0) Rat body weight 289 (14) 326 (19) 323(26) 325 (15) 327 (17) (Day 60) Rat body weight 319 (18) 359 (23) 360(36) 353 (18) 355 (29) (Day 180) Weight gain (%) 24.5^(a) 47.5^(b)50.3^(b) 46^(b) 45.4^(b) Mean food consumption  12.7 (0.73)  12.9 (0.34) 12.7 (0.82)  12.7 (0.72) 13.3 (1.4) (g/day) Values were recordedweekly. Weight gain represents the increased rate of weigh from thebeginning to the end of the experiment. Values are represented as mean(SD). One-way ANOVA: p < 0.05. Letters represents Fisher-LSD post hoccomparison tests.

A micro-CT (μCT) three-dimensional schematic microarchitecturereconstruction of a representative distal femur and 4th vertebra fromeach group is presented in FIG. 5. The reconstruction of the femoraltrabecular metaphysis as well as the vertebral body enablesdifferentiation of three distinct groups: the first group comprising theSham with the highest observed trabecular bone region (FIG. 5A), thesecond group comprising the OVX-CCC with the lowest trabecular boneregion (FIG. 5B) in comparison to all other groups, a third groupcomprising OVX-ACC and OVX-CCC+ALN with a modest increase in trabecularbone region in comparison to the CCC (FIGS. 5C and 5D) and a fourthgroup comprising OVX-ACC+ALN with an additive increase in trabecularbone region compared to the third group (FIG. 5E).

The differences between the four groups observed are also reflected inthe trabecular bone mineral density (BMD) and in the morphometricanalysis of the femur metaphysis and 4^(th) lumbar vertebra presented inFIG. 6 and Table 18.

As illustrated in FIG. 6A-B, the trabecular femoral BMD of the Shamgroup was significantly higher compared to all four OVX groups. Withinthe OVX groups, the trabecular femoral and vertebral BMD of the ACCtreated group (FIGS. 6A-B) were higher in comparison to the trabecularBMD of the CCC by 22% and 39%, respectively. The trabecular femoral andvertebral BMD of the CCC+ALN treated group were higher in comparison tothe trabecular BMD of the CCC by 49% and 72%, respectively. Thetrabecular femoral and vertebral BMD of the ACC+ALN treated group werehigher in comparison to the trabecular BMD of the CCC by 75% and 105%,respectively, and the addition of ACC to ALN instead of CCC increasedthe BMD by 26% and 33% in the femur and vertebra respectively,representing an additive positive effect on BMD values for ACC incombination with ALN.

In contrast to the trabecular BMD, no differences between all groupswere observed in the cortical bone BMD (Ct. BMD) of both the femur andvertebra (Table 18).

TABLE 18 Morphometric microarchitectural analysis obtained by μCTscanning OVX Sham CCC ACC CCC + ALN ACC + ALN (A) Tb. N, mm⁻¹ 4.1 (0.7)1.5 (0.3) 1.7 (0.1) 2.3 (0.3) 2.5 (0.3) Femur Tb. Sp, μm 167 (41) 682(93) 584 (95) 360 (64) 323 (47) Tb. Th, μm 111 (9) 110 (10) 112 (7) 102(11) 105 (7) Ct. BMD, g/cm³ 750 (32) 756 (30) 750 (67) 741 (58) 771 (27)TBPf, mm⁻¹ −7.0 (6.4) 7.6 (1.7) 6.1 (1.0) 7.3 (1.8) 6.3 (1.2) SMI 0.12(0.2) 1.66 (0.2) 1.50 (0.1) 1.63 (0.2) 1.55 (0.1) (B) Tb. N, mm⁻¹ 3.60(0.7) 1.75 (0.5) 2.05 (0.7) 2.54 (0.3) 2.73 (0.3) Vertebra Tb. Sp, μm199 (38) 374 (87) 352 (101) 268 (42) 251 (37) Tb. Th, μm 110 (17) 98.4(5.3) 107 (13) 100 (11) 109 (13) Ct. BMD, g/cm³ 750 (32) 756 (30) 750(67) 741 (58) 771 (27) TBPf, mm⁻¹ −0.7 (5.8) 8.5 (3.2) 6.8 (2.3) 4.8(1.8) 4.4 (1.9) SMI 0.61 (0.8) 1.62 (0.3) 1.56 (0.2) 1.28 (0.2) 1.22(0.3) (A) Distal femurs metaphysis. (B) 4^(th) lumbar vertebras. Tb. Nrepresents number of trabeculas within mm⁻¹ area, Tb. Sp represents themean distance between trabeculas, Tb. Th represents mean thickness ofthe trabeculas, TBPf represents trabecular bone pattern factor and SMIrepresents structural model index. Values are represented as mean (SD.

Morphometric analysis of the distal femur metaphysis and 4^(th)vertebral body exhibited a significant decrease in the trabecular bonefraction (BV/TV) in all ovariectomized groups in comparison to the Sham.Within the femurs and the vertebras of the OVX groups, ACC presented asignificantly higher trabecular bone fraction in comparison to the CCCgroup by 34% and 23%, respectively. Similar results were also obtainedfor CCC+ALN group, which presented an increase of 37% in the femur and46% in the vertebra in comparison to CCC. ACC in combination with ALNresulted in an increase of 56% in the femur and 71% in the vertebra incomparison to CCC, and in an increase of 19% and 25% in the femur andvertebra, respectively, in comparison to ALN+CCC group, againrepresenting an additive effect on increase in BV/TV values for ACC incombination with ALN (FIG. 6C-D).

A significant decrease in the trabecular number (Tb.N) in allovariectomized groups in comparison to the Sham was also observed inboth the femur and vertebra. Within the femurs and vertebras of the OVXgroups, ACC presented a higher trabecular number in comparison to theCCC group by an average of 17%. Higher results were obtained for CCC+ALNgroup, which presented an increase of 50% on average, in the femurvertebra in comparison to CCC. ACC in combination with ALN resulted inan average increase of 62% in the femur and vertebra in comparison toCCC, and in an average increase of 13% in the femur and vertebra, incomparison to ALN+CCC group, representing an additive effect on increasein Tb.N values for ACC in combination with ALN (Table 18).

A significant increase in the trabecular separation (Tb.Sp) in allovariectomized groups in comparison to the Sham was observed in both thefemur and vertebra. Within the femurs and vertebras of the OVX groups,ACC presented lower separation number in comparison to the CCC group byan average of 11%. Lower results were obtained for CCC+ALN group, whichpresented a decrease of 38% on average, in the femur and vertebra incomparison to CCC. ACC in combination with ALN resulted in a similardecrease of 54% in the femur and vertebra in comparison to CCC, and inan averaged decrease of 16% in the femur and vertebra, in comparison toALN+CCC group, further supporting the additive effect of ACC incombination with ALN (Table 18).

Analysis of the trabecular thickness (Tb.Th) reveled no changes amongall study groups in the femur, and only slight changes in the vertebra,manifested by an increase of only 12% in trabecular thickness in theSham group compared to CCC, an increase of 9% in the ACC group comparedto CCC group, only 2% increase following ALN treatment in comparison toCCC alone and a small additive effect of ACC in combination with ALNreflected by an increase of 11% in Tb.Th values, similar to the Shamgroup (Table 18).

Microarchitectural analysis of topology and orientation parameters:trabecular bone pattern factor (TBPf) and structural model index (SMI)revealed that ovariectomy intervention resulted in an increase in TBPfand SMI values in both femur and vertebra. In the femur ACC treatmentresulted in a greater decrease in TBPf and SMI values than CCC+ALN whichhad only a slight effect on these parameters in this region. Yet, in thevertebra, ACC treatment resulted in a smaller decrease in TBPf and SMIvalues compared to CCC+ALN. ACC+ALN treatment resulted in an additiveaveraged decrease of 12% in the femur and 37% in the vertebra for TBPfand SMI values in comparison to CCC treatment (Table 18).

Experimental Details Animals

Fifty three 16 weeks old female Sprague Dawley rats (Harlan Inc.,Jerusalem, Israel) with an average body weight of 246±17 g weregroup-housed in 26 cages (2-3 rats/cage) and acclimated under controlledroom conditions (21±2° C. and 12-hours dark-light cycle). During 7 daysacclimation, rats were fed with standard laboratory diet containing 1%elemental calcium (#2018SC Rat chow, Harlan Inc. Jerusalem, Israel) andde-ionized water ad-libitum.

For the administration of the different calcium sources, special foodpellets were prepared. Low calcium rodent pellets containing 0.02%calcium (Rat chow #TD95027 Harlan Inc., Jerusalem, Israel) was finelygrounded and separately mixed with two different calcium sources. A CCCmix containing 1% elemental calcium from crystalline calcium carbonate(Socal® Precipitated Calcium Carbonate), and an ACC mix containing 1%elemental calcium from stable amorphous calcium carbonate (containing 1%phosphoserine and 2% Citrate; Amorphical Ltd., Beer-Sheva, Israel).Every mix was separately pelleted and kept in a sealed bag at roomtemperature.

Following the 7 days acclimation period, rats were anesthetized byintraperitoneal (IP) injection of anesthetic solution (0.1 ml/100 g)containing Ketamine (Fort Dodge AH Ltd. Overland Park, USA) and Xylazine(EuroVet AH Ltd. Bladel, UK).

Rats were randomly assigned and operated according to their assignedfive groups: Sham (sham-ovariectomized; n=9), and four other groups wereovariectomized (OVX) by a bilateral dorsal approach according to Lasotaand Danowska-Klonowska (2004) Rocz Akad Med Bialymst 49 Suppl 1:129-131,and divided to OVX-CCC (n=10), OVX-ACC (n=10), OVX-CCC+ALN (n=12) andOVX-ACC+ALN (n=12). Following operation, rats were provided a 3 daysrecovery period through which they were treated with de-ionized watercontaining 1.5 ml/400 ml Dipyrone (Vitamed Ltd, Binyamina, Israel), and1 mg/kg Enrofloxacin (Buyer Ltd. Leverkusen, Germany). The animals wereleft untreated under the same conditions as the acclimation period,standard laboratory diet ad-libitum for 2 months to develop Osteopenia.

Following 2 months, rats started treatment according to their assignmentas described in Table 16 for a period of 4 months. The assignedsupplemental pellets and de-ionized water were provided ad-libitumduring the entire treatment period. Food consumption and body weightwere recorded weekly (Table 17). At the end of the treatment period, allrats were sacrificed by CO₂. Right femurs and 4th lumbar vertebras weredissected, wrapped with gaze pads soaked with saline buffer and kept in−80° C. for measurement of bone micro-CT scan.

Micro-Computed Tomography (pCT) Scanning

Trabecular and cortical bone microarchitecture of right femurs and4^(th) lumbar vertebras were analyzed in a μCT scanner (Skyscan 1174,Kartuizersweg Kontich, Belgium). Calibration of the scanner for bonemineral density (BMD) was performed according to manufactureinstructions using a designated rat phantom rod with densities of 0.25and 0.75 g/cm³ (Skyscan, Kartuizersweg Kontich, Belgium).

Prior to scan analysis, femurs and 4th lumbar vertebras were removedfrom −80° C. and placed in a plastic (according to the manufactureguidance). The distal region of each femur was scanned for trabecularand cortical bone parameters at an isotropic resolution of 13.8 μm.Reconstruction was carried out employing a modified algorithm using theSkyscan Nrecon software (ver. 1.6.4, Skyscan). Volume of interest (VOI)was set to 0.8 mm below growth plate, and extended distally for 1.5 mm.Trabecular and cortical segments within the VOI were extracted bydepicting ellipsoid contours every ˜5 slices using the CTan analysissoftware provided with the scanner (ver. 1.11 Skyscan). Trabecular andcortical BMD, bone volume (BV/TV, %), trabecular number (Tb.N),trabecular thickness (Tb.Th, μm), trabecular separation (Tb.Sp, μm)along with the micro-architectural parameters of trabecular bone patternfactor (TBPf) and structure model index (SMI) were calculatedautomatically by the CT analyzer software (ver. 1.11 Skyscan).

The 4^(th) lumbar vertebra from each rat was scanned in a 13.8 μmisotropic resolution and VOI was set to cover 1.25 mm thick crosssection from the center of the vertebral body for evaluation of thetrabecular and cortical parameters in the same procedure as describedabove for the femurs.

The results of the above trial confirmed that stable ACC has highereffect on bone mineral density and other bone parameters that CCC.Moreover, the administration of ACC in combination with standardmedication for treatment of bone loss resulted in additive effectcompared to CCC, allowing the decrease of bisphosphonate dosage andconfirming that highly bioavailable ACC can be used for increasing bonemineral density and treating metabolic bone disorders, diseases andconditions alone or together with the conservative medications.

Trial 7: Preventing or Delaying the Onset of Metabolic Bone Disorders

The inventors have conducted a study aiming to evaluate the effectcalcium from stable Amorphous Calcium Carbonate (ACC) compared tocrystalline calcium carbonate (CCC), on the prevention of bone metabolicdisorders and specifically osteoporosis as presented in theovariectomized (OVX) rat model.

In the study, five groups of rats were tested (4 ovariectomized and 1sham) The OVX animals were fed with special food mixtures in the form ofpellets from four different calcium sources: commercial crystallinecalcium carbonate served as control (mix A), natural ACC (Gastrolithpowder) (mix B), synthetic ACC (comprising trace amounts ofphospho-serine) (mix C), and commercial crystalline calcium citrate (mixD). Sham group was administrated with the same food as the control.Pellets from all four mixtures contained ˜0.5% elemental calcium asconfirmed by atomic absorption apparatus (Varian AA240, Palo Alto,Calif., USA) and described in Table 19 with no added vitamin D, exceptfor the amounts found in the original food pellets (22 IU vitamin D/g).

TABLE 19 Elemental calcium content (% in weight) of the calcium sourceand specialized pellets as was measured by atomic absorption CarbonateGastrolith ACC Citrate (Mix A) (Mix B) (Mix C) (Mix D) % Ca²⁺ in thecalcium source 34.54 28.1 33.0 18.3 % Ca²⁺ in the pellets 0.54 0.54 0.530.55

The source of the calcium was obtained from: commercial crystallineCaCO₃ (Carbonate), gastrolith powder from the Australian crayfish Cheraxquadricarinatus (Gastrolith), synthetic stable amorphous calciumcarbonate (ACC) and calcium citrate from a commercial supplement(Citrate). Values are represented as mean.

Table 20 presents the mean food consumption and body weight of the ratsduring the trial. A significant weight increase was observed in all OVXrats compared to sham. This is despite the fact that food consumptionwas similar among all groups.

TABLE 20 Mean food consumption and total body weight of the rats at thebeginning and at the end of the experiment OVX Sham Control GastrolithACC Citrate Rat body weight 253 (3.5) 249 (3.3) 249 (5.2) 251 (4)   253(4.7) (Day 0) Rat body weight 310 (8.3) 345 (5.8) 370 (6.4) 359 (4.2)354 (5.6) (Day 90) Weight gain 22.5^(a) 38.5^(b) 48.5^(b) 43^(b) 40^(b)(%) Mean food  15.2 (0.07)  15.2 (0.07)  15.2 (0.07)  15.1 (0.02)  15.2(0.05) consumption Values were recorded weekly. Weight gain representsthe increased rate of weigh from the beginning to the end of theexperiment. Values are represented as mean (SD). One-way ANOVA: p <0.05. Letters represents Fisher-LSD post hoc comparison tests.

A μCT three-dimensional schematic microarchitecture reconstruction of arepresentative distal femur and 4th vertebra from each group ispresented in FIG. 7. The reconstruction of the femoral trabecularmetaphysis as well as the vertebral body enables differentiation ofthree distinct groups: the first group comprising the Sham with thehighest observed trabecular bone region (FIG. 7A), the second groupcomprising the OVX Control and Citrate with the lowest trabecular boneregion (FIGS. 7B and 7E) in comparison to the Sham, and a third groupcomprising OVX Gast and ACC with a modest decrease in trabecular boneregion in comparison to the Sham (FIGS. 7C and 7D). The differencesbetween the three groups observed are also reflected in the trabecularbone mineral density (BMD) and in the morphometric analysis of the femurmetaphysis and 4^(th) lumbar vertebra presented in FIG. 8, FIG. 9 andTable 21.

As illustrated in FIG. 8A, the trabecular femoral BMD of the Sham groupwas significantly higher compared to all four OVX groups: the Control,Gast, ACC and Citrate with significant differences of 64%, 45%, 50% and63%, respectively. Comparable values were also obtained in thetrabecular vertebral BMD, presented in FIG. 8B, of the Sham groupcompared to all four OVX groups. Within the OVX groups, the trabecularfemoral and vertebral BMD of Gast and ACC (FIGS. 8A and 8B) weresignificantly higher in comparison to the trabecular BMD of the Controlby 56% and 38% in the femur and 38% and 32% in the vertebra,respectively. No significant differences were observed in the trabecularfemoral and vertebral BMD of the Citrate in comparison to the Control.Comparison between the Gast and ACC groups revealed statisticaldifferences of 13% in favor of Gast over the ACC in the trabecularfemoral BMD and no difference in the trabecular vertebral BMD.

In contrast to the trabecular BMD, no significant differences betweenall groups were observed in the cortical bone BMD (Ct. BMD) of both thefemur and vertebra (Table 21).

TABLE 21 Morphometric analysis and cortical bone mineral density(Ct.BMD) obtained by μCT scanning OVX Sham Control Gastrolith ACCCitrate (A) **Tb. N, mm⁻¹ 6.1^(a) (0.9) 2.2^(b) (0.6) 3.5^(c) (0.6)3.3^(c) (0.5) 2.4^(b) (0.5) Femur **Tb. Sp, μm 111^(a) (28) 248^(b) (83)158^(c) (46) 209^(d) (74) 226^(bd) (47) *Tb. Th, μm 31.5^(ac) (5.3)29.0^(b) (0.8) 33.6^(a) (3.5) 30.9^(cb) (1.4) 29.1^(b) (2.0) Ct. BMD,963 (51) 980 (57) 983 (62) 952 (53) 970 (53) g/cm³ (B) **Tb. N, mm⁻¹3.98^(a) (0.67) 1.57^(b) (0.33) 2.29^(c) (0.25) 2.24^(c) (0.34) 1.59^(b)(0.22) Vertebra **Tb. Sp, μm 153^(a) (28) 254^(b) (74) 197^(c) (54)229^(bc) (36) 262^(b) (60) Tb. Th, μm 84.6 (7.3) 87.5 (6.4) 83.8 (20.3)93.5 (6.9) 86.6 (8.5) Ct. BMD, 933 (55) 950 (50) 959 (56) 961 (57) 943(68) g/cm³ (A) Distal femurs metaphysis. (B) 4^(th) lumbar vertebras.Tb. N represents number of trabeculas within mm⁻¹ area, Tb. Sprepresents the mean distance between trabeculas, Tb. Th represents meanthickness of the trabeculas. One-way ANOVA: *p < 0.05, **p < 0.01.Letters represents Fisher-LSD post hoc comparison tests.

Morphometric analysis of the distal femur metaphysis and 4^(th)vertebral body (FIG. 9 and Table 21) exhibited a significant decrease inthe trabecular bone fraction (BV/TV) and its number (Tb.N) in allovariectomized groups in comparison to the Sham. The Control group wassignificantly lower by 65% on average in the distal femur (FIG. 9A andTable 21A) and by 60% in the vertebra (FIG. 3B and Table 3B) incomparison to the Sham. Whereas the average of the OVX Gast and ACCgroups in the distal femur was lower by only 41% and 49% in comparisonto the Sham, and lower in 40% and 41% in the vertebra, respectively.Within the femurs of the OVX groups, Gast and ACC presented asignificantly higher trabecular bone fraction and trabecular number incomparison to the Control by an average of 65% and 49%, respectively.Similar results were also obtained in the lumbar vertebra of the Gastand ACC groups with a higher average of 48% and 47% in comparison toControl, respectively. No significant differences were observed in thetrabecular bone fraction and its number between the Citrate and theControl.

Comparison between the femurs of Gast and ACC groups revealed asignificant difference in bone fraction, trabecular thickness andtrabecular separation. According to the results presented in FIG. 9A andTable 21A, the femurs of Gast group exhibited a significant higher bonefraction of 16%, a higher trabecular thickness of 9% and a lowertrabecular separation of 24% in comparison to ACC. A significantlyhigher trabecular thickness of 12% was also observed in the vertebra ofGast in comparison to ACC but no significant differences were observedin the rest of the morphometric parameters between Gast and ACC (FIG. 9Band Table 21B).

The differences between the groups in the microarchitecture structureare further emphasized in the un-decalcified structuralhistomorphometric analysis of the proximal tibia, presented in Table22A. According to the results presented in Table 22A, all OVX groups,namely the Control, Gast, ACC and Citrate presented significantly lowertrabecular bone fraction and condensed trabeculas values compared to theSham (with an average of 55%, 19%, 32% and 55%, respectively). Moreover,the trabecular separation was significantly higher in all OVX groups incomparison to Sham (Table 22A) by 130% for the Control, 30% for Gast,60% for ACC and 148% for Citrate. Within the OVX groups, Gast and ACCpresented a significantly higher trabecular bone fraction and trabecularnumber with an average of 80% and 50%, and a lower trabecular separationof 44% and 31%, in comparison to Control, respectively. The Citrategroup presented a higher significant trabecular thickness of 13%compared to Control. Comparison of structural histomorphometricparameters between Gast and ACC groups revealed a significant differencein bone fraction, trabecular number and trabecular separation.

According to Table 22A, Gast group presented higher bone fraction andtrabecular number of 19% and 21%, in comparison to ACC and a lowertrabecular separation value of 20% in comparison to ACC, respectively.

TABLE 22 Histomorphometric analysis of un-decalcified sections from theproximal tibias OVX Sham Control Gastrolith ACC Citrate (A) **BV/TV, %28.5^(a) (4.1) 12.8^(b) (2.1) 23^(c) (3.3) 19.4^(d) (2.8) 12.8^(b) (1.4)Structural **Tb. N, mm⁻¹ 5.6^(a) (0.6) 2.5^(b) (0.5) 4.5^(c) (0.4)3.7^(d) (0.8) 2.3^(b) (0.3) **Tb. Sp, μm 128^(a) (25) 296^(b) (44)166^(c) (15) 205^(d) (43) 318^(b) (42) *Tb. Th, μm 51.2^(ab) (6.2)47.1^(a) (6.0) 51.6^(ab) (7.1) 54.8^(b) (7.7) 53^(b) (9.9) (B) **BFR/BS,μm²/μm/day 0.145^(a) (0.026) 0.164^(a) (0.021) 0.214^(b) (0.025)0.206^(b) (0.027) 0.159^(a) (0.019) Dynamic **MS/BS, % 8.04^(a) (1.53)8.31^(a) (1.42) 11.61^(b) (1.36) 11.09^(b) (1.18) 8.25^(a) (1.44) MAR,μm/day 1.26 (0.26) 1.30 (0.19) 1.34 (0.25) 1.29 (0.21) 1.40 (0.20) (A)Structural parameters were analyzed from 4 μm stained sections(MacNeal). (B) Dynamic parameters were analyzed from un-decalcifiedsections double-labeled with calcein fluorescent dye. All values werecalculated using the BIOQUANT Image software analysis (Bioquant OSTEO,ver. 7.20.10). One-way ANOVA: *p < 0.05, **p < 0.01, Letters representsFisher-LSD post hoc comparison tests.

The results of the dynamic histomorphometry analysis are presented inTable 22B. According to the results, no significant differences wereobserved in the bone formation rate to bone surface (BFR/BS) andmineralizing surface to bone surface (MS/BS) of the OVX Control andCitrate groups in comparison to Sham. On the other hand, bone formationrate of the OVX Gast and ACC were 53% and 50% higher, in comparison toSham, and MS/BS of the OVX Gast and ACC presented similar elevations of44% and 38%, respectively compared to the sham. Within the OVX groups,Gast and ACC presented a significant higher bone formation rate of 38%and 35%, and significant higher MS/BS of 40% and 33%, in comparison toControl, respectively. The OVX Citrate group presented no significantdifference from to the Control but a statistical lower bone formationrate of 28% from Gast and 26% from ACC. MS/BS of the OVX Citrate groupwas similar to that of the Control and significantly lower by 29% and26% from the Gast and ACC groups, respectively.

Mineral apposition rate (MAR) revealed no significant differences in allOVX groups compared to Sham, and no differences of the OVX Gast, ACC andCitrate in comparison to Control. EIA kit assessment of bone turnover byserum osteocalcin (OC) formation marker and urine deoxypyridinoline(DPD) resorption marker of each group at the end of the experiment (day90) are presented in Table 23. Both markers were evaluated for baselineprior to randomization (at day 0, data not shown).

TABLE 23 Assessment of serum osteocalcin (OC) formation marker and urinedeoxypyridinoline (DPD) resorption marker for each group at day 90 OVXSham Control Gastrolith ACC Citrate *DPD 13.75^(a) 36.65^(be)29.823^(bd) 22.59^(ad) 43.55^(e) (nmol/mmol Cr) (5.44) (31.16) (16.56)(11.09) (22.97) OC (ng/ml) 10.0^(a) 14.8^(a) 14.72^(a) 12.7^(a) 15.7^(a)(4.8) (6.4) (8.7) (6.8) (8.4) One-way ANOVA: *p < 0.05

At day 90, significant higher DPD levels were observed in all OVX groupscompared to the Sham, except for the ACC group that presented anon-significant difference from the Sham, and a significant lower DPDlevel from the Control (39%), and Citrate (48%) groups. The Gast grouppresented lower level from the Control (19%) and higher DPD level fromthe ACC (32%) groups. DPD levels from Gast were not statistically lowerthan the Citrate but presented a difference of 31%. In the serum OCmarker, no significant changes were observed between any of the groups.

The results from the mechanical strength analysis of the 5^(th) lumbarvertebra are presented in FIG. 10. According to the results, themechanical strength properties of the OVX Control, Gast and Citrate weresignificantly lower in comparison to Sham by an average of 41%, 40% and40%, respectively. Whereas, the mechanical strength values of the ACCgroup were significantly lower by an average of 17% in comparison to theSham. These differences were statistically significant in mostparameters evaluated, except for the ultimate force parameter which wasnot statistically different (FIG. 10D). Within the OVX groups, ACC grouppresented significantly higher values in comparison to the Control inevery tested parameter: ultimate force (FIG. 10A), energy to ultimateforce (FIG. 10B), toughness (FIG. 10C) and energy to yield (FIG. 10D) byan average of 37%, 49%, 29% and 65%, respectively. These differences arealso reflected in the in the higher values of OVX ACC in comparison tothe Gast and Citrate groups with an average of 42% and 37%,respectively. The OVX Gast and Citrate groups did not present anystatistical difference to the Control or between them in any of themechanical strength parameters tested.

Micro-CT analysis of the 4^(th)-lumbar vertebra showed that thetrabecular bone pattern factor (TBPf) is far lower (91% on average) inthe sham group than in the OVX-control, OVX-gastrolith and OVX-citrategroups. The TBPf of the sham group was, however, lower (61%) than whatwas measured for the OVX-ACC group. Within the OVX groups, the TBPf ofthe OVX-ACC groups was significantly lower than that of the OVX-control,OVX-citrate and OVX-gastrolith groups by an average of 33% (FIG. 10E;p<0.001). Structural model index (SMI) evaluation points to the sametrend, being significantly lower in the sham group, compared to theOVX-control, OVX-gastrolith and OVX-citrate groups (43% on average). TheSMI of the sham group was, however, lower (30%) than what was measuredfor the OVX-ACC group. Here, values obtained with the OVX-ACC group weresignificantly lower that what was measured for the OVX-control andOVX-citrate groups (14% on average). At the same time, SMI valuesobtained with the OVX-ACC group were reduced relative to the indexmeasured with the OVX-gastrolith group (FIG. 10F; p<0.001). The lastmicro-architectural parameter measured, namely the degree of anisotropy(DA), was lower in the sham group, compared to the OVX-control,OVX-gastrolith and OVX-citrate groups, by 22% on average. No significantdifferences in DA was observed between the sham and OVX-ACC groups, oramong all OVX groups, although OVX-ACC showed lower values that measuredwith the other OVX groups (FIG. 10G; p<0.001).

Experimental Details Animals

One hundred and one 16-17 weeks old female Sprague Dawley rats (HarlanInc., Jerusalem, Israel) with an average body weight of 250±16 g weregroup-housed in 33 cages (˜3 rats/cage) and acclimated under controlledroom conditions (21±2° C. and 12-hours dark-light cycle). During 7 daysacclimation, rats were fed with standard laboratory diet containing 1%elemental calcium (#2018SC Rat chow, Harlan Inc. Jerusalem, Israel) andde-ionized water ad-libitum. Following the 7 days acclimation period,rats were housed individually in metabolic cages and deprived from foodand water for 18 h. Urine was collected from each metabolic funnel andwas stored at −80° C. for future analysis of bone resorption biochemicalmarker at baseline (time 0).

For the administration of the different calcium sources, special foodpellets containing were prepared. Low calcium rodent pellets containing0.02% calcium (Rat chow #TD95027 Harlan Inc., Jerusalem, Israel) wasfinely grounded and separately mixed with four different calciumsources. A control mix containing calcium carbonate from a commercialsupplement (mix A), Gast mix containing gastrolith powder harvested fromthe Australian crayfish Cherax quadricarinatus (mix B) (Ben's farm, BeerTzofar, Israel), ACC mix containing stable amorphous calcium carbonateand P-serine (P-Ser) (mix C) (Batch #ETS001, Amorphical Ltd.,Beer-sheva, Israel) and the Citrate mix containing calcium citrate froma commercial supplement (mix D). Every mix was separately pelleted andkept in a sealed bag at room temperature.

Immediately after urine sampling, rats were anesthetized usingintraperitoneal (IP) injection of anesthetic solution (0.1 ml/100 g)containing Ketamine (Fort Dodge AH Ltd. Overland Park, USA) and Xylazine(EuroVet AH Ltd. Bladel, UK). Blood was sampled from rat's tail(approximately 180 μl) and centrifuged for 10 minutes at 3000 g using atabletop centrifuge (Hettich Zentrifugen, Bach, Switzerland) for serumextraction. Serum samples were stored at −80° C. for future analysis ofbone formation biochemical marker at baseline (Time 0).

Rats were randomly assigned and operated according to their assignedfive groups: Sham (sham-Ovariectomized) fed with mix A, and four othergroups were ovariectomized (OVX) using a bilateral dorsal approachaccording to Lasota and Danowska-Klonowska (2004) Rocz Akad Med Bialymst49 Suppl 1:129-131, and divided to OVX Control (fed with mix A), OVXGast (fed with mix B), OVX ACC (fed with mix C) and OVX Citrate (fedwith mix D). Following operation, rats were provided a 7 days recoveryperiod under the same conditions as the acclimation period, standardlaboratory diet ad-libitum and de-ionized water containing 1.5 ml/400 mlDipyrone (Vitamed Ltd, Binyamina, Israel), and 1 mg/kg Enrofloxacin(Buyer Ltd. Leverkusen, Germany).

Experimental Treatment

At the end of the recovery period the rats were group-housed in 47 cages(2 rats/cage) according to the group assignment, under the samecontrolled room conditions as described above. The assigned supplementalpellets and de-ionized water were provided ad-libitum during the entiretreatment period (90 days). Food consumption and body weight wererecorded weekly (Table 2). For dynamic histomorphometric analysisfluorochrom dye Calcein (20 mg/kg body mass; Sigma-Aldrich, Israel) wasIP injected to all rats at days 76 and 86. On day 90, rats wereindividually housed in metabolic cages and deprived from food and waterfor 18 h. Urine was collected from each metabolic funnel and was storedat −80° C. for future analysis of bone resorption biochemical marker atend of the experiment (Time 90) Immediately after urine sampling, serumsamples were extracted for evaluation of formation biochemical marker ofday 90 and stored in the same manner as described at day 0. All ratswere sacrificed by CO₂. Right femurs and 4th lumbar vertebras weredissected, wrapped with gaze pads soaked with saline buffer and kept in−80° C. for measurement of bone micro-CT scan. The 5th lumbar vertebraswere stored in the same conditions as the right femurs for mechanicaltesting. Right tibias were dissected and placed immediately for 24 h at4° C. in 3.7% formaldehyde solution (F1636, Sigma-Aldrich, Israel) forstructural and dynamic histomorphometric measurements.

Micro-Computed Tomography (pCT) Scanning

Trabecular and cortical bone microarchitecture of right femurs and4^(th) lumbar vertebras were analyzed using a μCT scanner (Skyscan 1174,Kartuizersweg Kontich, Belgium). Calibration of the scanner for bonemineral density (BMD) was performed according to manufactureinstructions using a designated rat phantom rod with densities of 0.25and 0.75 g/cm³ (Skyscan, Kartuizersweg Kontich, Belgium).

Prior to scan analysis, femurs and 4th lumbar vertebras were removedfrom −80° C., transferred to new gauze pads soaked with 70% ethanol andplaced in a plastic vial filled with 70% ethanol (according to themanufacture guidance). The distal region of each femur was scanned fortrabecular and cortical bone parameters at an isotropic resolution of13.8 μm. Reconstruction was carried out employing a modified algorithmusing the Skyscan Nrecon software (ver. 1.6.4, Skyscan). Volume ofinterest (VOI) was set to 0.5 mm below growth plate, and extendeddistally for 1.5 mm. Trabecular and cortical segments within the VOIwere extracted by depicting ellipsoid contours every ˜5 slices using theCTan analysis software provided with the scanner (ver. 1.11 Skyscan).Trabecular and cortical BMD, bone volume (BV/TV, %), trabecular number(Tb.N), trabecular thickness (Tb.Th, μm), and trabecular separation(Tb.Sp, μm) were calculated automatically by the CTan analyzer software(ver. 1.11 Skyscan).

The 4^(th) lumbar vertebra from each rat was scanned in a 13.8 μmisotropic resolution and VOI was set to cover 2.0 mm thick cross sectionfrom the center of the vertebral body. Trabecular and cortical boneparameters were evaluated in the vertebral body, set to cover a VOI of a2-mm thick cross-section, starting 0.14 mm below the cranial growthplate. In addition to the same trabecular and cortical bone parametersas studied for the distal femur, the topology parameters: i.e,trabecular bone pattern factor (TBPf) and structural model index (SMI);and the trabecular orientation parameter, degree of anisotropy (DA) werealso measured in the vertebral body.

Histomorphometry

Right tibias were removed from the formaldehyde solution and dehydratedthrough an ethanol gradient, cleared using xylene (24250521, Bio-LabLtd. Haifa, Israel), and infiltrated with 80% methylmethacrylate (MMA;M5599, Sigma Aldrich, Rehovot, Israel)+20% dibutyl phthalate (DBP;61-5062, Merck Ltd, Hohenbrunn, Germany) for 3 days at 4° C. Specimenswere then transferred to MMA+DBP+Benozoyl peroxide (617008, Merck Ltd,Hohenbrunn, Germany) solution for another 6 days infiltration at 4° C.Following infiltration procedure, specimens were embedded inMMA+DBP+2.5% Benozoyl peroxide under a temperature gradient (from 38° C.to 50° C. over a period of 4 days). Mid-sagittal (4 μm) un-decalcifiedsections from each specimen were cut using a Lecia RM 2025 microtome(Lecia Instruments Ltd, Petach Tikva, Israel) connected to tungstencarbide knifes (Disposable blade; TC65, Rhenium Inc. Bet-Nekufa,Israel). Most sections were stained with McNeal's tetrachrome forstructural histomorphometry. The remaining sections were left unstainedfor dynamic histomorphometry.

Measurement of both structural and dynamic histomorphometry of theproximal tibia metaphisyes were obtained using a semi-automatic analysissystem (Bioquant OSTEO, ver. 7.20.10, BIOQUANT Image AnalysisCorporation Nashville, USA) which is connected to a microscope (NikonOptiphot 2, Kingston, England) equipped with fluorescent light source.

Histomorphometric parameters such as bone volume (BV/TV, %), trabecularnumber (Tb.N), trabecular thickness (Tb.Th, μm), trabecular separation(Tb.Sp, μm), bone formation rate/bone sample (BFR/BS), mineralizingsurface/bone surface (MS/BS) and mineral apposition rate (MAR) werecalculated using the BIOQUANT Image software analysis (Bioquant OSTEO,ver. 7.20.10).

Evaluation of Bone Turnover Biochemical Markers

Serum Osteocalcin (OC) concentration was determined by EIA kit (#BT-490,Biomedical Technologies Inc. Stoughtone, USA) following a 1:10 serumdilution.

Urinary Deoxypyridinoline (DPD) concentration was determined bycommercial EIA kit (#8007, Qudiel Inc. San Diego, USA) following a 1:50dilution. DPD levels were normalized to Creatinine levels (#8009, QudielInc. San Diego, USA).

Mechanical Strength of the 5th Lumbar Vertebra

Mechanical properties (Ultimate force, Energy to ultimate force,Toughness and Energy to yield) were obtained from the compression testsof 5th lumbar vertebral body (L5). Calculation of the materialproperties were performed by measuring the height of the vertebral bodyand the average of caudal and cranial diameters using a caliper. Crosssectional area (CSA) and bone volume (BV) were obtained by μCT scanningthat covered the entire bone including both trabecular and corticalshell.

Statistical Analysis

Results are expressed herein as means±SEM. One-way ANOVA and Fisher-LSDpost hoc comparison tests were performed using STATISTICA 6.1 software(StaSoft, Tulsa, Okla.). A p value<0.05 was deemed significant.

The results of the above trial confirmed that stable ACC has highereffect on bone mineral density and other bone parameters that CCC instudy group susceptible to the development of osteoporosis. The superiorbioavailability of ACC allows the use thereof for preventing or delayingthe onset of metabolic bone disorders, diseases and conditions.

Although embodiments of the invention have been described by way ofillustration, it will be understood that the invention may be carriedout with many variations, modifications, and adaptations, withoutexceeding the scope of the claims.

1. A method for enhancement of bone mineral density in a subjectsuffering from a bone metabolism associated disorder, disease, orcondition, the method comprising: administering to said subject aneffective amount of a composition including stable amorphous calciumcarbonate (ACC) having at least one stabilizer, in combination with abisphosphonate, wherein the bisphosphonate is administered in a doselower than a standard therapeutic dose.
 2. The method according to claim1, wherein said at least one stabilizer is selected from the groupconsisting of organic acids, phosphoric or sulfuric esters of hydroxycarboxylic acids, and hydroxyl bearing organic compounds.
 3. The methodaccording to claim 1, wherein the bisphosphonate is selected from thegroup consisting of Alendronate, Risedronate, Tiludronate, Ibandronate,Zolendronate, Pamidronate, Etidronate, salts thereof, and estersthereof.
 4. The method according to claim 3, wherein the bisphosphonateis Alendronate.
 5. The method according to claim 2, wherein said atleast one stabilizer includes at least one component selected from thegroup consisting of phosphoric esters of hydroxy carboxylic acids andphosphoric esters of hydroxyl bearing organic compounds.
 6. The methodaccording to claim 2, wherein said at least one stabilizer is a hydroxylbearing organic compound selected from the group consisting ofmono-saccharides, di-saccharides, tri-saccharides, oligo-saccharides,and poly-saccharides.
 7. The method according to claim 2, wherein saidat least one stabilizer includes hydroxyl bearing organic compounds,further combined with at least one alkali hydroxide.
 8. The methodaccording to claim 2, wherein said at least one stabilizer is acarboxylic acid or a plurality of carboxylic acids.
 9. The methodaccording to claim 8, wherein said one or more carboxylic acids includeat least one acid selected from the group consisting of citric acid,tartaric acid, and malic acid.
 10. The method according to claim 2,wherein said at least one stabilizer includes at least one compoundselected from the group consisting of phosphorylated amino acids,phosphorylated peptides, chitin together with at least one peptide, andpolyol together with alkaline hydroxide.
 11. The method according toclaim 2, wherein said at least one stabilizer is a phosphorylated aminoacid.
 12. The method according to claim 11, wherein the phosphorylatedamino acids are present in oligopeptides or polypeptides.
 13. The methodaccording to claim 11, wherein the phosphorylated amino acids areselected from the group consisting of phosphoserine andphosphothreonine.
 14. A method of increasing calcium gastrointestinal(GI) absorption in a subject susceptible to development of a bonemetabolism associated disorder, said method comprising: administering tosaid subject a composition including stable amorphous calcium carbonate(ACC) having at least one stabilizer, wherein the GI absorption ofcalcium from said composition is at least 1.9 times higher than from acorresponding composition comprising an equivalent dose of crystallinecalcium carbonate.
 15. The method according to claim 14, wherein thesubject is a postmenopausal woman.